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Ras-Activated Endocytosis Is Mediated by the Rab5 Guanine Nucleotide Exchange Activity of RIN1
Abstract
RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.
... Thus, EMT in glioma cells was inhibited, accompanied by suppression of cell migration and invasion. Furthermore, POSTN depletion promoted the internalization of EGFR and interactions between EGFR and Ras and Rab interactor 1 (RIN1), a protein required for EGFR endocytosis [17,18]. Collectively, these results indicate that POSTN/ EGFR/RIN1 interactions could potentially be leveraged as novel therapeutic targets in glioma, even in refractory cases. ...
... Compared with those in the corresponding siNC-transfected cells, the levels of EGFR at the perinuclear region were increased in POSTN-downregulated U87MG and U251MG cells (Fig. 5A). The study by Tall et al. showed that activation of the Rab5 GTPase by RIN1 is a major step in regulating EGFR endocytosis [17]. In addition, RIN1 localization to the plasma membrane determines the endocytic fate of EGFR [18]. ...
... As several studies have shown, activation of Rab5 plays a key role in the internalization and endosomal trafficking of EGFR [31][32][33], which is mediated by the Rab5 guanine nucleotide exchange factor RIN1 [17,34]. A possible model based on the current study is shown in Fig. 6. ...
Purpose
Approximately 1/3 of brain tumors are gliomas. Previous glioma-related studies have reported increased expression of periostin (POSTN) in these cancerous tissues, but the role and mechanism of POSTN in glioma development remain unclear.
Methods
Nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC–MS/MS) and RNA sequencing were used to identify differential protein and mRNA expression in clinical glioma samples. Quantitative real-time PCR (qRT–PCR) was used to measure the expression of POSTN in tissues and cells. The effects of POSTN on glioma cell migration and invasion were examined using wound healing, Transwell, and three-dimensional spheroid assays in vitro and a nude mouse xenograft model in vivo . The effects of POSTN on the stability, endocytosis, and degradation of EGFR were examined by immunoblotting and immunofluorescence staining. Truncation mutation analysis was performed to investigate direct interactions between POSTN and EGFR. Immunohistochemical staining was carried out to confirm the clinical significance of POSTN.
Results
Overexpression of POSTN induced epithelial-to-mesenchymal transition (EMT) in glioma cells in vivo and in vitro . Mechanistically, POSTN downregulation inhibited EGFR signaling by promoting EGFR endocytosis and degradation. In addition, POSTN was found to bind to EGFR and RIN1, inhibiting EGFR endocytosis and degradation and thus activating the PI3K-Akt signaling pathway.
Conclusion
These findings indicate the mechanism by which the POSTN/EGFR/RIN1 axis inhibits EGFR endocytosis and degradation, resulting in glioma cell EMT through the PI3K-AKT signaling pathway. Targeting POSTN/EGFR/RIN1 interactions may guarantee beneficial outcomes of glioma treatment.
... Amongst HRAS effectors the serine/threonine RAF kinases, the catalytic subunits of phosphoinositide 3-kinases (PIK3CA), phospholipase C1 (PLCE1) and RAL guanine nucleotide dissociation stimulator (RALGDS) are best characterized (4). Less is known about the HRAS effector RAS and RAB interactor 1 (RIN1) and its associated signaling pathways (5,6): binding of active HRAS to RIN1 promotes the activation of RAB5 GTPases (7) and ABL1/2 tyrosine kinases (8). Through these two signaling branches, endosomal sorting events and cytoskeletal dynamics are coordinated (7)(8)(9)(10)(11)(12). ...
... Less is known about the HRAS effector RAS and RAB interactor 1 (RIN1) and its associated signaling pathways (5,6): binding of active HRAS to RIN1 promotes the activation of RAB5 GTPases (7) and ABL1/2 tyrosine kinases (8). Through these two signaling branches, endosomal sorting events and cytoskeletal dynamics are coordinated (7)(8)(9)(10)(11)(12). Translocation of RIN1 between cytoplasmic and membrane compartments is controlled in part by Ser 351 phosphorylationdependent binding to 14-3-3 proteins (10,13). ...
... RIN1 is a guanine nucleotide exchange factor for the RASrelated protein RAB5A and an activator of non-receptor tyrosineprotein kinases ABL1/2 (7,8). To determine HRAS-RIN1 stimulated RAB5A activation, we measured the release of f luorescently labeled GDP (mant-deoxy-GDP) from recombinant RAB5A. ...
Heterozygous germline missense variants in the HRAS gene underlie Costello syndrome (CS). The molecular basis for cutaneous manifestations in CS is largely unknown. We used an immortalized human cell line, HaCaT keratinocytes, stably expressing wild-type or CS-associated (p.Gly12Ser) HRAS and defined RIN1 as quantitatively most prominent, high-affinity effector of active HRAS in these cells. As an exchange factor for RAB5 GTPases, RIN1 is involved in endosomal sorting of cell adhesion integrins. RIN1-dependent RAB5A activation was strongly increased by HRASGly12Ser, and HRAS-RIN1-ABL1/2 signaling was induced in HRASWT- and HRASGly12Ser-expressing cells. Along with that, HRASGly12Ser expression decreased total integrin levels and enriched β1 integrin in RAB5- and EEA1-positive early endosomes. The intracellular level of active β1 integrin was increased in HRASGly12Ser HaCaT keratinocytes due to impaired recycling, whereas RIN1 disruption raised β1 integrin cell surface distribution. HRASGly12Ser induced co-localization of β1 integrin with SNX17 and RAB7 in early/sorting and late endosomes, respectively. Thus, by retaining β1 integrin in intracellular endosomal compartments, HRAS-RIN1 signaling affects the subcellular availability of β1 integrin. This may interfere with integrin-dependent processes as we detected for HRASGly12Ser cells spreading on fibronectin. We conclude that dysregulation of receptor trafficking and integrin-dependent processes such as cell adhesion are relevant in the pathobiology of CS.
... It was since suggested that any proteins that contained the same modularity and biochemical characteristic of the Vps9 domain may also function as a GEF to activate Rab5 (Hama et al., 1999). Based on these principles, RIN1 (Tall et al., 2001), RIN2 (Saito et al., 2002), RIN3 (Kajiho et al., 2003b;Kajiho et al., 2011) and RINL (Woller et al., 2011;Kajiho et al., 2012), were all identified and confirmed to function as GEFs to promote the activation of Rab5. ...
... In particular, the Rabex5 VPS9 domain exhibited a potent GEF activity specific for Rab5, Rab21 and weak activity for Rab22, while showing no detectable activity for other Rab proteins (Delprato et al., 2004). Due to the presence of a VPS9 domain in RIN1, it was not surprising that RIN1 was shown to have strong GEF activity for Rab5A, Rab5B, Rab5C and Vps21p (Tall et al., 2001). Kajiho and colleagues employed elegant biochemistry studies and demonstrated that RIN3 also promoted GTP binding of Rab31, another member of the Rab5 family (Kajiho et al., 2003b;Kajiho et al., 2011). ...
... Evidence for their interaction or Ras induced RIN3 mediated endocytic alternation are still missing. Hypothetically, RIN3 might bind Ras via RA domain, and activated Ras might also promote Rab5 activation and endocytic receptor mediated internalization, which makes RIN3 another bridge for Ras-cell signaling and Rab5-endocytosis process (Tall et al., 2001). ...
Ras and Rab interactor 3 (RIN3) functions as a Guanine nucleotide Exchange Factor (GEF) for some members of the Rab family of small GTPase. By promoting the activation of Rab5, RIN3 plays an important role in regulating endocytosis and endocytic trafficking. In addition, RIN3 activates Ras, another small GTPase, that controls multiple signaling pathways to regulate cellular function. Increasing evidence suggests that dysregulation of RIN3 activity may contribute to the pathogenesis of several disease conditions ranging from Paget’s Disease of the Bone (PDB), Alzheimer’s Disease (AD), Chronic Obstructive Pulmonary Disease (COPD) and to obesity. Recent genome-wide association studies (GWAS) identified variants in the RIN3 gene to be linked with these disease conditions. Interestingly, some variants appear to be missense mutations in the functional domains of the RIN3 protein while most variants are located in the noncoding regions of the RIN3 gene, potentially altering its gene expression. However, neither the protein structure of RIN3 nor its exact function(s) (except for its GEF activity) has been fully defined. Furthermore, how the polymorphisms/variants contribute to disease pathogenesis remain to be understood. Herein, we examine, and review published studies in an attempt to provide a better understanding of the physiological function of RIN3; More importantly, we construct a framework linking the polymorphisms/variants of RIN3 to altered cell signaling and endocytic traffic, and to potential disease mechanism(s).
... Rab5 is primarily localised to early endosomes (Gorvel, Chavrier et al. 1991) and plays a role in membrane budding and trafficking in the early endocytic pathways (Kajiho, Saito et al. 2003;Saito, Kajiho et al. 2005;Kajiho, Sakurai et al. 2011;Woller, Luiskandl et al. 2011 (Han, Wong et al. 1997) (Colicelli, Nicolette et al. 1991;Saito, Murai et al. 2002). RIN2 and RIN3 preferentially interact with the GTP-bound form of Rab5, while RIN1 preferentially interacts with GDPbound Rab5 (Tall, Barbieri et al. 2001;Saito, Murai et al. 2002). ...
... The RIN1 RA domain was initially identified as a region interacting with HRAS (Hofer, Fields et al. 1994;Ponting and Benjamin 1996), suggesting that RINs are capable of binding not only to Rab5 but also to the RAS-family GTPases. The GEF activity of RIN1 for Rab5 is enhanced by its interaction with HRAS (Han and Colicelli 1995;Tall, Barbieri et al. 2001). However, HRAS does not interact with RIN3 or RIN2 to stimulate their GEF activities in conditions under which it certainly binds to RIN1 (Rodriguez-Viciana, Sabatier et al. 2004;Wohlgemuth, Kiel et al. 2005). ...
... In HeLa cells RIN3 and RIN2 localise with Rab5 to endocytic vesicles, whereas RIN1 exhibits a cytoplasmic distribution. RIN1 partially co-localises with Rab5-positive vesicles upon co-expression, and stimulates epidermal-growth-factor-receptor (EGFR)-mediated endocytosis (Tall, Barbieri et al. 2001). ...
Classical RAS oncogenes are mutated in approximately 30% of human tumours and RAP proteins are closely related to classical RAS proteins. RAP1 has an identical effector domain to RAS whereas RAP2 differs by one amino acid. RAP2 not only shares effectors with other classical RAS family members, but it also has its own specific effectors that do not bind to RAP1 or classical RAS family proteins. Thus, although closely related, RAP2 performs distinct functions, although these have been poorly characterised. Using RAP2 as bait in Tandem Affinity Purifications, we have identified several RAP2 interacting proteins including TRIM32; a protein implicated in diverse pathological processes such as Limb-Girdle Muscular Dystrophy (LGMD2H), and Bardet-Biedl syndrome (BBS). TRIM32 was shown to interact specifically with RAP2 in an activation- and effector domain-dependent manner; demonstrating stronger interaction with the RAP2 V12 mutant than the wild-type RAP2 and defective binding to the effector mutant RAP2 V12A38. The interaction was mapped to the C-terminus of TRIM32 (containing the NHL domains) while mutations found in LGMD2H (R394H, D487N, Δ588) were found to disrupt binding to RAP2. The TRIM32 P130S mutant linked to BBS did not affect binding to RAP2, suggesting that the RAP2-TRIM32 interaction may be functionally involved in LGMD2H. Because TRIM32 is an E3 ubiquitin ligase, the possible ubiquitination of interacting proteins by TRIM32 was assessed along with the potential for modulation by RAP2. RAP2 stimulates the ubiquitin ligase activity of TRIM32 against some substrates but not others. We propose that RAP2 uses TRIM32 to regulate the signalling properties of other RAP2 effectors. Furthermore, our data also shows that the overexpression of TRIM32 may increase C2C12 mouse myoblast cell differentiation whereas the inhibition of RAP2 expression decreases differentiation in C2C12 cells. Further study could lead to a potential link to Limb-Girdle Muscular Dystrophy that remains to be elucidated.
... RIN1 functions through two downstream pathways involved in the maintenance of epithelial properties namely the activation of Rab5 [11] and ABL family tyrosine kinases [8]. By activating ABL tyrosine kinases, RIN1 blocks the cytoskeletal rearrangements associated with cell dissociation and migration. ...
... By activating ABL tyrosine kinases, RIN1 blocks the cytoskeletal rearrangements associated with cell dissociation and migration. RIN1 is a multidomain Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking [11]. RIN1 is thus a Ras effector protein with its Ras binding domain (RBD) localized in its carboxyl-terminal region. ...
Background
Head and neck tumors (HNT) are tumors of the paranasal sinuses, the salivary glands and the upper aerodigestive tract. RIN1 is a Ras effector protein regulating epithelial cell properties and has been implicated in a number of cancers.
Method
The aim of this study was to investigate the expression of RIN1 in head and neck tumors. RIN1 expression was assessed using quantitative real-time PCR (qRT-PCR) and immunohistochemical staining on archival head and neck tissue samples between 2014 and 2020.
Results
RIN1 expression was low in tissue samples as compared with the normal head and neck tissues. High and low RIN1 levels were compared with ages ≤40, >40 in the head and neck tumors of p-value 0.02. There was a significant difference with p-values of 0.001 when poor and well-moderate malignant tumors were compared.
Conclusion
Our data suggests that RIN1may play an important role in head and neck tumor progression and that its expression may provide baseline data to facilitate identification of new molecular targeted therapeutics.
... In addition to its ability to moderate mitogenic signaling through the Ras/MAPK pathway, RIN1 also acts as a guanine nucleotide exchange factor (GEF) for the small monomeric GTPase known as Rab5 (Tall et al., 2001). The Rab proteins are a diverse group of proteins that belong to the Ras superfamily of small GTPases and play critical roles in regulating the steps of endocytic vesicular transport. ...
... Rab5, which is activated by RIN1, has been shown to regulate vesicle budding and cargo selection from clathrincoated pits as well as early endosome fusion (Stein et al., 2003;Hutagalung and Novick, 2011;Stenmark and Olkkonen, 2001). Tall et al. (2001) report that RIN1 and Rab5 also play a crucial role in the receptor-mediated endocytosis of epidermal growth factor (EGF)-receptor, following stimulation by EGF. The endocytosis of EGFreceptor occurs through a Ras-mediated mechanism involving upstream activation of RIN1 by GTP-Ras and then subsequent activation of Rab5 by activated RIN1. ...
... No reuse allowed without permission. Figure S1). Figure 1d lists the top 40 predictions: 18 of the 40 predictions (orange and blue boxes) were previously established as KRAS modulators 32-34 or KRAS effectors [35][36][37][38][39][40][41][42][43][44][45] . Green text identifies proteins that were observed to physically interact with KRAS 46,47 . ...
... No reuse allowed without permission. 44 , proteins predicted by PrePPI to interact with KRAS are more likely to also interact with RAB5A (ρ = 0.47). While only PrePPI predictions for KRASspecific protein-protein interactions were included to avoid this potential issue, other similar subtle dependencies may be harder to identify and address. ...
The largely incomplete and tissue-independent nature of cancer pathways represents a key limitation to the ability to elucidate mechanistic determinants of cancer phenotypes and to predict adaptive response to targeted therapy. To address these challenges, we propose replacing canonical cancer pathways with a more accurate, comprehensive, and context-specific architecture – dubbed a Protein-Centric molecular interaction Map (PC-Map) – representing modulators, effectors, and cognate binding-partners of any oncoprotein of interest. To reconstruct these complex molecular architectures de novo , we introduce a novel OncoSig algorithm. Validation of a lung adenocarcinoma specific (LUAD) KRAS-centric PC-Map recapitulated known KRAS biology and, more critically, identified a novel repertoire of proteins eliciting synthetic lethality in KRAS G12D LUAD organoid cultures. Showing the generalizable nature of the algorithm, we elucidated PC-Maps for ten recurrently mutated oncoproteins, including KRAS, in distinct tumor contexts. This revealed a highly context-specific nature of cancer’s regulatory and signaling architectures to an unprecedented degree of resolution.
... Furthermore, also the expression o proteins involved in intracellular vesicle ormation and tracking (e.g. DYNC1I1, DYNC1I2, ERC1, EXOC7, PRRC2C, RAB23, RIN1, RRAS, SNX1, TOM1L2, TSG101, VAPA) [53][54][55][56][57][58][59] was modied. A gene ontology-based analysis o the pSILAC results allowed us to dene the cellular unctions o proteins whose expression was aected by CBDA treatment (Fig. 6C and D). ...
Eukaryotic Initiation Translation Factor 2A (EIF2A) is considered to be primarily responsible for the initiation of translation when a cell is subjected to stressful conditions. However, information regarding this protein is still incomplete. Using a combination of proteomic approaches, we demonstrated that EIF2A is the molecular target of the naturally occurring bioactive compound cannabidiolic acid (CBDA) within human glioblastoma cells. This finding allowed us to undertake a study aimed at obtaining further information on the functions that EIF2A plays in tumor cells. Indeed, our data showed that CBDA is able to activate EIF2A when the cells are in no-stress conditions. It induces conformational changes in the protein structure, thus increasing EIF2A affinity towards the proteins participating in the Eukaryotic Translation Machinery. Consequently, following glioblastoma cells incubation with CBDA we observed an enhanced neosynthesis of proteins involved in the stress response, nucleic acid translation and organization, and protein catabolism. These changes in gene expression resulted in increased levels of ubiquitinated proteins and accumulation of the autophagosome. Our results, in addition to shedding light on the molecular mechanism underlying the biological effect of a phytocannabinoid in cancer cells, demonstrated that EIF2A plays a critical role in regulation of protein homeostasis.
... On the contrary, the lifetime of active Ras was significantly prolonged on macropinosomes in the absence of Rab5A (figure 5b). In mammalian cells, active Ras recruits and activates Rab5 on the macropinosomes by interacting with Rab5 GEF RIN1 [27,75,80]. Ras activation peaks almost concurrently with the maximal localization of Rab5, and Ras leaves Rab5-positive macropinosome, possibly via a fast recycling route, before fusion with other endocytic vesicles [75,77]. ...
RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A–IqgC interaction remains elusive.
... They were described as GEFs for Rab5 sharing besides the Vps9 domain, also Src homology 2 (SH2), proline-rich (PR), RIN family homology (RH), and Ras association (RA) domains. The clinical outcome of deficiencies or overexpression in Rin1-3 range from neurological conditions like Alzheimer's disease to macrocephaly, alopecia, cutis laxa, and scoliosis syndrome (Kajiho et al., 2003;Saito et al., 2002;Tall et al., 2001). The latest member of the Rin protein family, Rinl, was originally identified as an interaction partner of the muscle-specific receptor tyrosine kinase MuSK. ...
T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4⁺ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4⁺ T cells unraveled T cell–intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4⁺ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4⁺ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.
... Relative levels of PI3K, AKT, SGK1, JNK, ERK, mTOR, p38, and RAF-1 were determined using the ratio of p-PI3K, p-AKT, p-SGK1, p-JNK, p-ERK, p-mTOR, p-p38, p-RAF-1 to t-PI3K, t-AKT, t-SGK1, t-JNK, t-ERK, t-mTOR, t-p38, and t-RAF-1, respectively. Relative levels of survivin, IAP-1, IAP-2, Cyclin-D1, COX-2, VEGF, Caspase, PARP, p53, IGF-1R, and p21 proteins were determined by densitometry using the ratio of these proteins to GAPDH as previously described (Tall et al., 2001). ...
Backgorund
Fruits and seed extracts of Annona montana have significant cytotoxic potential in several cancer cells. This study evaluates the effect of A. montana leaves hexane extract on several signaling cascades and gene expression in metastatic breast cancer cells upon insulin-like growth factor-1 (IGF-1) stimulation.
Methods
MTT assay was performed to determine the proliferation of cancer cells. Propidium iodide staining and flow cytometry analysis of Annexin V binding was utilized to measure the progression of the cell cycle and the induction of apoptosis. Protein expression and phosphorylation were determined by western blotting analysis to examine the underlying cellular mechanism triggered upon treatment with A. montana leaves hexane extract.
Results
A. montana leaves hexane (sub-fraction V) blocked the constitutive stimulation of the PI3K/mTOR signaling pathways. This inhibitory effect was associated with apoptosis induction as evidenced by the positivity with Annexin V and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNNEL) staining, activation of caspase-3, and cleavage of PPAR. It also limited the expression of various downstream genes that regulate proliferation, survival, metastasis, and angiogenesis (i.e., cyclin D1, survivin, COX-2, and VEGF). It increased the expression of p53 and p21. Interestingly, we also observed that this extract blocked the activation of AKT and ERK without affecting the phosphorylation of the IGF-1 receptor and activation of Ras upon IGF-1 stimulation.
Conclusion
Our study indicates that A. montana leaves (sub-fraction V) extract exhibits a selective anti-proliferative and proapoptotic effect on the metastatic MDA-MB-231 breast cancer cells through the involvement of PI3K/AKT/mTOR/S6K1 pathways.
... Therefore, GEFs are vital for Rabs to maintain active, and several GEFs for Rab21 have been identified, including VPS9-ankyrin-repeat protein (Varp) [19], Rabaptin-5-associated exchange factor for Rab5 (Rabex5) [20,21], SET-binding factor (Sbf) [22], and Legionella pneumophila family guanine-nucleotide exchange effector 0393 (Lpg0393) [23]. Besides, although Rab21 and Rab5 are derived from the same family in the phylogenetic tree and both belong to the Rab5 subfamily [3,24], and all the Ras interaction (RIN) family proteins contain a vacuolar protein-sorting (VPS) domain and function as Rab5-GEF [25][26][27], no RIN family members show significant GEF activity for Rab21 [28]. ...
Rab proteins are important components of small GTPases and play crucial roles in regulating intracellular transportation and cargo delivery. Maintaining the proper functions of Rab proteins is essential for normal cellular activities such as cell signaling, division, and survival. Due to their vital and irreplaceable role in regulating intracellular vesicle transportation, accumulated researches have shown that the abnormalities of Rab proteins and their effectors are closely related to human diseases. Here, this review focused on Rab21, a member of the Rab family, and introduced the structures and functions of Rab21, as well as the regulatory mechanisms of Rab21 in human diseases, including neurodegenerative diseases, cancer, and inflammation. In summary, we described in detail the role of Rab21 in human diseases and provide insights into the potential of Rab21 as a therapeutic target for diseases.
... Ras and Rab interactor 1 (Rin1) is a specific GEF that enhances Rab5 activity and induces Ras/MEK/ERK signalling. 9 We found that C20orf24-WT, but not C20orf24ΔR, enhanced the interaction between Rab5 and Rin1, indicating that the Rab5ip domain of C20orf24 is essential for Rab5 to recruit Rin1 ( Figure 3L). Interestingly, an interaction between C20orf24 and Rin1 was observed, which could be reduced when Rab5ip domain was deleted ( Figure S5C), suggesting that Rab5ip domain of C20orf24 is critical for its binding to Rin1. ...
... 8 Overexpression of wild-type (WT) or a constitutively activated (Q79L) mutant Rab5 in cancer cells maximally stimulates endocytosis, promoting b1 integrin internalization and cell migration. 8 In line with this idea, increased expression of Rab5 GEFs, such as RIN1, 13 and decreased expression of its GAPs, such as RN-tre, 14 both increase Rab5 activity and are related to metastasis in cancer patients. GEF/ GAP-regulated Rab5 is a potential therapeutic target in cancer; however, the regulatory mechanisms behind the actions of GAPs and GEFs on the activity of Rab5 are not well understood. ...
Endocytosis of cell surface receptors is essential for cell migration and cancer metastasis. Rab5, a small GTPases of Rab family, is a key regulator of endosome dynamics and thus cell migration, however, how its activity is regulated yet remains to be addressed. Here, we identified a Rab5 inhibitor, a long non-coding RNA namely HITT (HIF-1α inhibitor at translation level). Our data show that HITT expression is inversely associated with advanced stages and poorly prognosis of lung adenocarcinoma patients with area under receiver operating characteristics (ROC) curve (AUC) 0.6473. Further study reveals that both endogenous and exogenous HITT inhibits single cell migration by repressing β1 integrin endocytosis in lung adenocarcinoma. Mechanistically, HITT is physically associated with Rab5 at switch I via 1248–1347nt and suppresses β1 integrin endocytosis and subsequent cancer metastasis by interfering with guanine nucleotide exchange factors (GEFs) for Rab5 binding. Collectively, these findings suggest that HITT directly participates in the regulation of Rab5 activity, leading to a decreased integrin internalization and cancer metastasis, which provides important insights into a mechanistic understanding of endocytosis and cancer metastasis.
... Rab5 and the GTPase Dynamin regulate CME (Zeigerer et al., 2012) and, downstream of actomyosin contractility, act to remove membrane excess (Lee and Harland, 2010). Additionally, Rab5 has also been implicated in macropinocytosis (Tall et al., 2001;Barbieri et al., 2004;Lanzetti et al., 2004). ...
Morphogenesis in early embryos demands the coordinated distribution of cells and tissues to their final destination in a spatio-temporal controlled way. Spatial and scalar differences in adhesion and contractility are essential for these morphogenetic movements, while the role that membrane remodeling may play remains less clear. To evaluate how membrane turnover modulates tissue arrangements we studied the role of endocytosis in zebrafish epiboly. Experimental analyses and modeling have shown that the expansion of the blastoderm relies on an asymmetry of mechanical tension in the yolk cell generated as a result of actomyosin-dependent contraction and membrane removal. Here we show that the GTPase Rab5ab is essential for the endocytosis and the removal of the external yolk cell syncytial layer (E-YSL) membrane. Interfering in its expression exclusively in the yolk resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal flows, a disequilibrium in force balance and epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics, preserving embryo geometry and coordinating morphogenetic movements during epiboly.
... The 29 proteins are involved in different stages of the endocytosis pathway. For example, APLP1, DNM1, and PAK1 regulate the endocytic uptake of extracellular materials (Gammie et al., 1995;Neumann et al., 2006;Karjalainen et al., 2008;Grassart et al., 2010); INPP5F, CMTM3, RIN1, and RIN3 participate in early endosome maturation (Tall et al., 2001;Kajiho et al., 2003;Yoshikawa et al., 2008;Galvis et al., 2009;Yuan et al., 2017;Taefehshokr et al., 2021); CLIC4, CTSS, LAPTM4B, SYTL4, TMEM106B, S100A9, and TOM1L1 maintain the normal function of late endosomes (Munger et al., 1995;Puertollano, 2005;Ostrowski et al., 2010;Brady et al., 2013;Blom et al., 2015;Tsai et al., 2015;Hsu et al., 2019); NSG1, RAB11FIP4, RAB15, S100A10, RAB13, and RAP2A might mediate endosome recycling back to the cell surface (Zuk and Elferink, 2000;Wallace et al., 2002;Zobiack et al., 2003;Morimoto et al., 2005;Taguchi and Misaki, 2011; Parkinson and Hanley, 2018); GOLIM4, RAB29, SGSM1, and ARL4A are involved in endosome to Golgi trafficking (Natarajan and Linstedt, 2004;Lin et al., 2011;Nottingham et al., 2012;Inoshita and Imai, 2015); CORO1C and KIF5A regulate endosome fission and endosome transport in the cytosol, respectively, (Schmidt et al., 2009;Hoyer et al., 2018); VAV2 and ZFYVE28 are able to divert EGFR endosomal trafficking and lysosomal degradation, although the detailed molecular mechanisms are elusive (Mosesson et al., 2009;Thalappilly et al., 2010). The risk score was calculated for each patient based on the mRNA expression levels and risk coefficients of the 29 selected genes. ...
Background
Low-grade glioma (LGG) is a heterogeneous tumor that might develop into high-grade malignant glioma, which markedly reduces patient survival time. Endocytosis is a cellular process responsible for the internalization of cell surface proteins or external materials into the cytosol. Dysregulated endocytic pathways have been linked to all steps of oncogenesis, from initial transformation to late invasion and metastasis. However, endocytosis-related gene (ERG) signatures have not been used to study the correlations between endocytosis and prognosis in cancer. Therefore, it is essential to develop a prognostic model for LGG based on the expression profiles of ERGs.
Methods
The Cancer Genome Atlas and the Genotype-Tissue Expression database were used to identify differentially expressed ERGs in LGG patients. Gene ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene set enrichment analysis methodologies were adopted for functional analysis. A protein-protein interaction (PPI) network was constructed and hub genes were identified based on the Search Tool for the Retrieval of Interacting Proteins database. Univariate and multivariate Cox regression analyses were used to develop an ERG signature to predict the overall survival (OS) of LGG patients. Finally, the association between the ERG signature and gene mutation status was further analyzed.
Results
Sixty-two ERGs showed distinct mRNA expression patterns between normal brain tissues and LGG tissues. Functional analysis indicated that these ERGs were strikingly enriched in endosomal trafficking pathways. The PPI network indicated that EGFR was the most central protein. We then built a 29-gene signature, dividing patients into high-risk and low-risk groups with significantly different OS times. The prognostic performance of the 29-gene signature was validated in another LGG cohort. Additionally, we found that the mutation scores calculated based on the TTN, PIK3CA, NF1, and IDH1 mutation status were significantly correlated with the endocytosis-related prognostic signature. Finally, a clinical nomogram with a concordance index of 0.881 predicted the survival probability of LGG patients by integrating clinicopathologic features and ERG signatures.
Conclusion
Our ERG-based prediction models could serve as an independent prognostic tool to accurately predict the outcomes of LGG.
... In melanoma cells, oncogenic class I PI3K elicits a hyperactive influx of macropinosomes, which is counteracted by Rab7A (Alonso-Curbelo et al., 2015). Furthermore, by stimulating RIN1, which is a Rab5 GEF, activation of H-Ras or H-Ras G12V also mediates homotypic fusion of early endosomes, thus leading to endosome enlargement (Roberts et al., 2000;Tall et al., 2001). ...
The volumes of a cell [cell volume (CV)] and its organelles are adjusted by osmoregulatory processes. During pinocytosis, extracellular fluid volume equivalent to its CV is incorporated within an hour and membrane area equivalent to the cell’s surface within 30 min. Since neither fluid uptake nor membrane consumption leads to swelling or shrinkage, cells must be equipped with potent volume regulatory mechanisms. Normally, cells respond to outwardly or inwardly directed osmotic gradients by a volume decrease and increase, respectively, i.e., they shrink or swell but then try to recover their CV. However, when a cell death (CD) pathway is triggered, CV persistently decreases in isotonic conditions in apoptosis and it increases in necrosis. One type of CD associated with cell swelling is due to a dysfunctional pinocytosis. Methuosis, a non-apoptotic CD phenotype, occurs when cells accumulate too much fluid by macropinocytosis. In contrast to functional pinocytosis, in methuosis, macropinosomes neither recycle nor fuse with lysosomes but with each other to form giant vacuoles, which finally cause rupture of the plasma membrane (PM). Understanding methuosis longs for the understanding of the ionic mechanisms of cell volume regulation (CVR) and vesicular volume regulation (VVR). In nascent macropinosomes, ion channels and transporters are derived from the PM. Along trafficking from the PM to the perinuclear area, the equipment of channels and transporters of the vesicle membrane changes by retrieval, addition, and recycling from and back to the PM, causing profound changes in vesicular ion concentrations, acidification, and—most importantly—shrinkage of the macropinosome, which is indispensable for its proper targeting and cargo processing. In this review, we discuss ion and water transport mechanisms with respect to CVR and VVR and with special emphasis on pinocytosis and methuosis. We describe various aspects of the complex mutual interplay between extracellular and intracellular ions and ion gradients, the PM and vesicular membrane, phosphoinositides, monomeric G proteins and their targets, as well as the submembranous cytoskeleton. Our aim is to highlight important cellular mechanisms, components, and processes that may lead to methuotic CD upon their derangement.
... A vast network of interaction partners of Rab5 has been identified, providing Rab5 with one of the most complex interactomes among the Rab family (Christoforidis et al., 1999). This includes GEFs like Rabex-5 and RIN1 (Horiuchi et al., 1997;Tall et al., 2001), but also Rab5specific GAPs such as RN-Tre (Lanzetti et al., 2000) and Rab-GAP5 (Haas et al., 2005). Within the large pool of downstream effectors, early endosome antigen 1 (EEA1) is probably the best RNA transport serves as a prime example of how spatio-temporal control can influence the expression of genes, underlying essential biological processes such as embryonic development or neuronal plasticity (Medioni et al., 2012;Mofatteh, 2020). ...
Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction is linked to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and the early endosome in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, in which two arm-like appendages, each consisting of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. We demonstrate that the coiled-coil domains of Fy-2 are flexible and project into opposite directions from the FERRY complex core. While the C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils together with Fy-5 bind mRNA. Thus, Fy-2 serves as binding hub that connects not only all five complex subunits, but also mediates the binding to mRNA and to the early endosome via Rab5. The FERRY structure provides novel mechanistic insight into long-distance mRNA transport.
... Besides Raf-1 and PI3K, the best-characterized effectors are members of a family of exchange factors for the small GTPase Ral, e.g., RalGDS, which in human cells is directly involved in oncogenesis [28]. Another known effectors are phosphoinositide-specific phospholipase C epsilon (PLCε), T lymphoma invasion and metastasis protein 1 [Tiam 1] and Rac, RAS association domain family (RASSF) [30], RIN1 [31], AF6 (Afadin) [32]and PKCζ [33]. ...
Many different regulatory mechanisms of renal fibrosis are known to date, and those related to transforming growth factor-β1 (TGF-β1)-induced signaling have been studied in greater depth. However, in recent years, other signaling pathways have been identified, which contribute to the regulation of these pathological processes. Several studies by our team and others have revealed the involvement of small Ras GTPases in the regulation of the cellular processes that occur in renal fibrosis, such as the activation and proliferation of myofibroblasts or the accumulation of extracellular matrix (ECM) proteins. Intracellular signaling mediated by TGF-β1 and Ras GTPases are closely related, and this interaction also occurs during the development of renal fibrosis. In this review, we update the available in vitro and in vivo knowledge on the role of Ras and its main effectors, such as Erk and Akt, in the cellular mechanisms that occur during the regulation of kidney fibrosis (ECM synthesis, accumulation and activation of myofibroblasts, apoptosis and survival of tubular epithelial cells), as well as the therapeutic strategies for targeting the Ras pathway to intervene on the development of renal fibrosis.
... DEGs from endocytosis pathway were also enriched in 27 out of 32 plant species roots exposed to graphene (Fig. 4). Endocytosis requires the Hsp70 protein family genes 31,32 and Rab (Ras-related protein) genes, which is a key regulator of cellular endocytosis 33,34 . These two endocytosis-related genes were selected to investigate their expression variation after graphene treatment. ...
To explore the effects of graphene on plant root growth and development, 25 mg/L graphene were used to treat the seedling roots of 48 plant species. These results showed that the total root length of the plants was decreased when cultured by hydroponics method. Whereas the total root length of plant species cultured by soil showed different growth effects, among which the ratio of promotion effect was 69.77%, the ratio of inhibition effect was 11.63% and the no-effect ratio was 18.60%. To gain insights into the molecular mechanisms by which graphene presents different growth effects on the root length, we performed RNA-seq for 32 plant roots treated with 25 mg/L graphene. We totally identified 90,259 DEGs in 32 plant species, among which 55,537 were graphene-induced and 34,722 were graphene-repressed. KEGG pathway enrichment analysis indicated that 43 pathways were assigned to these enriched differentially expressed genes in response to the graphene treatment. Top enriched pathways include starch and sucrose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, the citrate cycle (TCA cycle), phenylpropanoid biosynthesis, glutathione metabolism, endocytosis, peroxisome etc. The gene expressions of these pathway were induced or repressive in plant roots showing promotion or inhibitory effects, respectively. Accumulation of antioxidant enzyme as well as enhanced respiration might lead to the increasing plant root length. In addition, transcriptome and TEM data showed that graphene enters plant root cells by endocytosis. These results uncovered molecular level influences of graphene on plant roots development.
... Macropinocytosis is both Rac1-and actin-dependent and many studies have linked it to the ability to form membrane folds. Importantly, Rab5 (usually implicated in CME [5]) and its effector rabankyrin 5 are also involved in macropinocytosis [6][7][8]. Whatever their origin, vesicle trafficking through the endosomal route is controlled by specific Rab guanosine triphosphatases (GTPases): ...
Morphogenetic processes demand the coordinated allocation of cells and tissues to their final destination in a spatio-temporal controlled way. Identifying how these morphogenetic movements are directed and implemented is essential for understanding morphogenesis. Topographical and scalar differences in adhesion and contractility within and between cells are essential, yet, the role that membrane remodeling may play remains less clear. To clarify how surface turnover and dynamics may modulate tissue arrangements we studied epiboly in the zebrafish. During epiboly the blastoderm expands as a result of an asymmetry of mechanical tension along the embryo surface. In this scenario, we found that the membrane removal by macropinocytosis of the external yolk cell syncytial layer (E-YSL) ahead of the blastoderm is key for epiboly progression In early zebrafish embryos, the activity of the GTPase Rab5ab was essential for endocytosis, and interference in its expression exclusively in the yolk cell resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal yolk flows, a disequilibrium in force balance and as a result epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics and coordinating morphogenetic movements during epiboly.
... Other Rab5 GEFs that localize and recruit Rab5 to different intracellular compartments (e.g. GAPVD1 or RIN1 on clathrin-coated vesicles and the plasma membrane; Tall et al., 2001;Semerdjieva et al., 2008 have as of yet not been found to be coupled to effector activity. In vivo Rabex5/Rabaptin5 can be targeted to the EE by interaction of Rabex5 with ubiquitinated receptors and the binding of Ubiquitin to Rabex5 enhances nucleotide exchange activity (Lee et al., 2006;Mattera et al., 2006;Penengo et al., 2006;Lauer et al., 2019). ...
Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a ‘handover’ of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.
... Other Rab5 GEFs that localize and recruit Rab5 to different intracellular compartments (e.g. GAPVD1 or RIN1 on clathrin-coated vesicles and the plasma membrane; Tall et al., 2001;Semerdjieva et al., 2008 have as of yet not been found to be coupled to effector activity. In vivo Rabex5/Rabaptin5 can be targeted to the EE by interaction of Rabex5 with ubiquitinated receptors and the binding of Ubiquitin to Rabex5 enhances nucleotide exchange activity (Lee et al., 2006;Mattera et al., 2006;Penengo et al., 2006;Lauer et al., 2019). ...
Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a ‘handover’ of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.
... Other Rab5 GEFs that localize and recruit Rab5 to different intracellular compartments (e.g. GAPVD1 or RIN1 on clathrin-coated vesicles and the plasma membrane; Tall et al., 2001;Semerdjieva et al., 2008 have as of yet not been found to be coupled to effector activity. In vivo Rabex5/Rabaptin5 can be targeted to the EE by interaction of Rabex5 with ubiquitinated receptors and the binding of Ubiquitin to Rabex5 enhances nucleotide exchange activity (Lee et al., 2006;Mattera et al., 2006;Penengo et al., 2006;Lauer et al., 2019). ...
Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a ‘handover’ of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.
... EGF receptor endocytosis is popular model to study regulation of cargo traffic along degradative pathway. EGF binding to its receptor (EGFR) stimulates the receptor tyrosine kinase (TK) and one of the first consequences is recruitment onto membrane of the endosomal vesicle and activation of RIN1, Rab5-specific GEF, immediately upon internalization [17,18]. In fact, EGF/EGFR complex drives its own endocytosis by initiating certain molecular events, among which Rab5 activation is the key one. ...
Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins’ level and is sensitive to biosynthetic, but not endocytic pathway disturbance. EEA1-vesicles directly fuse with incoming EGF/EGFR-vesicles into hybrid endosomes with separated EEA1- and EGFR-domains, thus providing a platform for rapid achievement of an excess of surface-derived membrane that is used to form intraluminal vesicles (ILVs). Thus, multivesicular structures colocalized with EEA1 are still early endosomes. “EEA1-cycle” ends by exclusion of EGFR-containing domains with ILVs inside that turns into MVE and restoration of initial EEA1-vesicles population.
... https://doi.org/10.1101/2020.04.20.051623 doi: bioRxiv preprint RIN1 was previously shown to bind HRAS in vitro (20), and functions as a nucleotide exchange factor for RAB5 GTPases, which are central organizers of endocytosis (21). Recent work shows KRAS-transformed cancers use macropinocytosis, a specific form of endocytosis, to scavenge nutrients from their environment (22,23). ...
Activating mutations in RAS GTPases drive one fifth of cancers, but poor understanding of many RAS effectors and regulators, and of the roles of their different paralogs, continues to impede drug development. We developed a multi-stage discovery and screening process to understand RAS function and identify RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry (AP/MS), we generated a protein-protein interaction map of the RAS pathway containing thousands of interactions. From this network we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for genetic interactions (GIs). We found important new effectors of RAS-driven cellular functions, RADIL and the GEF RIN1, and over 250 synthetic lethal GIs, including a potent KRAS -dependent interaction between RAP1GDS1 and RHOA . Many GIs link specific paralogs within and between gene families. These findings illustrate the power of the multiomic approach to identify synthetic lethal combinations for hitherto undruggable cancers.
STATEMENT OF SIGNIFICANCE
We present a thorough survey of protein-protein and genetic interactions in the Ras pathway. These interactions suggested new discoveries that we validate here, and demonstrate important new paralog specificities and redundancies. By comparing synthetic lethal interactions across KRAS -dependent and -independent tumors, we identify new combination therapy targets against Ras-driven cancers.
... Therefore, the results show that Egfr signaling is also required for the vesicle-mediated release of ecdysone from PG cells. Interestingly, direct effects of Egfr signaling on the endocytic machinery have been already described in Drosophila tracheal cells as well as in human cells [26][27][28][29][30][31][32][33]. ...
Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow [1]. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult [2]. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3) [3, 4, 5, 6]. It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG [7, 8]. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition [9, 10, 11] mandates for a reconsideration of the conventional model. Here, we show that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. We found that Egfr signaling is activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size.
... 28 Tall et al's animal model study confirmed that statins have a potential effect on mitochondrial membrane through inhibition of carnitine palmitoyl transferase leading to the reduction of the attachment of Ras-family protein (Rab5) which is involved in neuronal endocytosis. 29 Accumulating evidence supports a key role for extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the development of the central nervous system (CNS) and in the regulation of peripheral neuron functions. ERK1/2, is one of the most well characterized members of the mitogen-activated protein kinase family, regulates a range of processes, from metabolism, motility and inflammation, to cell death and survival. ...
Statins are hydroxymethylglutaryl-coenzyme A reductase inhibitors inhibit denovo cholesterol synthesis leading to reduction of serum cholesterol and low density lipoprotein as well as elevation of high density lipoprotein level. Statins are used in the treatment of dyslipidaemia, prevention of major cardiovascular events and complications. The potential role of statins in the induction of peripheral neuropathy has not been verified as most of statins induced-peripheral neuropathy had been reported as case reports. Also, statins therapy leads to noteworthy reduction of Coenzyme Q10, causing impairment of neuronal energy. The incidence of polyneuropathy was high with atorvastatin (65%) which is lipophilic, and relatively less with fluvastatin (54%) which is hydrophilic. Long-term statins therapy, mainly with atorvastatin and simvastatin, is linked with the development of peripheral neuropathy.
... Therefore, the results show that Egfr signaling is also required for the vesicle-mediated release of ecdysone from PG cells. Interestingly, direct effects of Egfr signaling on the endocytic machinery have been already described in Drosophila tracheal cells as well as in human cells [26][27][28][29][30][31][32][33]. ...
... Of special interest is rin1a; orthologous to mammalian Rin1 (Rab and Ras interactor protein). In mammals, Rin1 can directly interact with Ras and a membrane trafficking GTPase Rab5, regulating receptor-dependent endocytosis (436). Furthermore, Rin1 and Raf-1 actually compete for binding of Ras, and possibly the signal transduction of Ras is determined by the competition between Rin1 and Raf-1 (437). ...
... Based on protein structure and homology models described in the literature and SWISS-MODEL protein structure homology modelling [30], the p.K689R variant is predicted to be located in the vicinity of the end of the αHB4 helix of the helical bundle of the RH domain. Deletion of this homologous region in a splice variant of RIN1 prevents its binding with Rab5 [31,32]. The p.Y793H, p.K838T and p.R859C variants are predicted to be located in the αV4, αV6 and αC helices of the VPS9 domain. ...
Paget’s disease of bone (PDB) is a common, late-onset bone disorder characterized by focal increase of bone turnover. Mutations in the SQSTM1 gene are found in up to 40% of patients and recent GWAS have led to novel associations with several loci. RIN3, the candidate gene located at the associated 14q32 locus, has recently been studied in a British cohort to elucidate its contribution to the pathogenesis. In this study, we performed a genetic screening of RIN3 in an unrelated cohort to validate these findings and to further explore genetic variation in this gene in the context of PDB. In our screening, we examined the 5′ untranslated region (UTR), the exonic regions and the intron–exon boundaries of the gene in a control cohort and a patient cohort. Our findings show clustering of variation similar to the British cohort and support a protective role for common genetic variation (rs117068593, p.R279C) in the proline-rich region and a functionally relevant role for rare genetic variation in the domains that mediate binding and activation of its interaction partner, Rab5. Additive regression models, fitted for the common variants, validated the association of the rs117068593 variant with the disease (OR+/+ 0.315; OR+/− 0.562). In addition, our analyses revealed a potentially modifying effect of this variant on the age of onset of the disease. In conclusion, our findings support the involvement of genetic variation in RIN3 in PDB and suggest a role for RIN3 as a potential modifier of the age of onset of the disease.
... RIN1 is a cytosol-localized specific GEF for RAB5/vacuolar protein sorting-associated protein 21 (VPS21) with RA domain and involved in RAS-regulated endocytosis [72]. RIN2 and RIN3 have been detected in endocytic vesicles. ...
Cellular activation of RAS GTPases into the GTP-binding “ON” state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson’s disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets.
... Mammalian Rab5 and Rab7, Rab family small GTPases, regulate early endosome and late endosome, respectively [96]. Rab5 promotes EGFR internalization, while Rab7 regulates EGFR trafficking from late endosomes to lysosomes [97][98][99][100][101]. The ortholog of mammalian Rab7, C. elegans RAB-7 was shown to be a negative regulator of LET-23: the rab-7 mutant suppressed the vulvaless phenotype of let-23(sy1) and lin-2(e1309) [102]. ...
EGF, emitted by the Anchor Cell, patterns six equipotent C. elegans vulval precursor cells to assume a precise array of three cell fates with high fidelity. A group of core and modulatory signaling cascades forms a signaling network that demonstrates plasticity during the transition from naïve to terminally differentiated cells. In this review, we summarize the history of classical developmental manipulations and molecular genetics experiments that led to our understanding of the signals governing this process, and discuss principles of signal transduction and developmental biology that have emerged from these studies.
... The observation of disrupted endosomal pathways in DS would be consistent with previous reports of enlarged early endosomes and increased endocytic uptake in Tri21 models (71,72,112,113). Rab5 is critical for endocytosis, endosomal sorting (74) and it also participates in autophagosome formation and closure (73,(114)(115)(116)(117)(118)(119). Our work regarding early endosomes reveals no difference in induction of Rab5 . ...
Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21 (Tri21). This unbalanced karyotype has the ability to produce proteotoxic stress and dysfunction of the proteostasis network (PN), which are mechanistically associated with several comorbidities found in the DS phenotype. Autophagy is the cellular process responsible for bulk protein degradation and its impairment could negatively impact protein quality control. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts. Both euploid (CTL) and DS fibroblasts induced autophagy successfully through serum starvation (SS), as evidenced by increased LC3-II abundance in CTL and DS. However, DS cells displayed evidence of autophagolysosome (AL) accumulation and impaired clearance of autophagosome cargo, e.g. accumulation of p62 and NBR1. Similar observations were also present in DS cells from multiple differentiation stages, implicating impeded autophagic degradation as a possible early pathologic mechanism in DS. Lysosomal pH and cathepsin B proteolytic activity were found to not differ in CTL and DS fibroblasts after SS, indicating that lysosomal dysfunction did not appear to contribute to unsuccessful autophagic clearance. Based on these results, we hypothesized that possible interference of the endosomal system with autophagy results in autophagosome fusion with endosomal vesicles and negatively impacts degradation. Consistent with this hypothesis, we observed increased abundance of the recycling endosome marker, Rab11, after SS in DS fibroblasts. Further, colocalization of autophagosome markers with resident proteins of early endosomes, late endosomes and recycling endosomes (Rab11) further support our hypothesis. In summary, our work is consistent with impairment of autophagic flux and general PN dysfunction as candidate mechanisms for pathology in DS.
... Hence, inhibition or overexpression of the ubiquitin ligase Rabex5, which is responsible for H-and NRas ubiquitination on endosomes, resulted in increased or attenuated Raf1/MAPK activation, respectively (Xu et al. 2010;Yan et al. 2010). In a much more complex scenario, Rin1, the GEF for Rab5, can bind HRas on endosomes and simultaneously stimulates Rab5-dependent endocytosis Tall et al. 2001). If the latter then triggers Rabex5 recruitment, this could also lead to HRas ubiquitination, thereby generating a negative feedback mechanism for HRas/MAPK activation. ...
The endocytic compartment is not only the functional continuity of the plasma membrane but consists of a diverse collection of intracellular heterogeneous complex structures that transport, amplify, sustain, and/or sort signaling molecules. Over the years, it has become evident that early, late, and recycling endosomes represent an interconnected vesicular-tubular network able to form signaling platforms that dynamically and efficiently translate extracellular signals into biological outcome. Cell activation, differentiation, migration, death, and survival are some of the endpoints of endosomal signaling. Hence, to understand the role of the endosomal system in signal transduction in space and time, it is therefore necessary to dissect and identify the plethora of decoders that are operational in the different steps along the endocytic pathway. In this chapter, we focus on the regulation of spatiotemporal signaling in cells, considering endosomes as central platforms, in which several small GTPases proteins of the Ras superfamily, in particular Ras and Rac1, actively participate to control cellular processes like proliferation and cell mobility.
... Among three isoforms of RIN, RIN1 is cytosolic while RIN2 and RIN3 are localized to endocytic vesicles. RIN1 with RA domain functions as GEF for RAB5/VPS21 like proteins and promotes RAS mediated endocytosis (Tall et al. 2001). Phospholipase C (PLC), RAS binding protein, gets activated by PDGF and EGF growth factors in a RAS and RAP1 dependent manner and has a critical role in carcinogenesis (Song et al. 2002). ...
RAS effector signaling instead of being simple, unidirectional and linear cascade, is actually recognized as highly complex and dynamic signaling network. RAF-MEK-ERK cascade, being at the center of complex signaling network, links to multiple scaffold proteins through feed forward and feedback mechanisms and dynamically regulate tumor initiation and progression. Three isoforms of Ras harbor mutations in a cell and tissue specific manner. Besides mutations, their epigenetic silencing also attributes them to exhibit oncogenic activities. Recent evidences support the functions of RAS oncoproteins in the acquisition of tumor cells with Epithelial-to-mesenchymal transition (EMT) features/ epithelial plasticity, enhanced metastatic potential and poor patient survival. Google Scholar electronic databases and PubMed were searched for original papers and reviews available till date to collect information on stimulation of EMT core inducers in a Ras driven cancer and their regulation in metastatic spread. Improved understanding of the mechanistic basis of regulatory interactions of microRNAs (miRs) and EMT by reprogramming the expression of targets in Ras activated cancer, may help in designing effective anticancer therapies. Apparent lack of adverse events associated with the delivery of miRs and tissue response make ‘drug target miRNA’ an ideal therapeutic tool to achieve progression free clinical response.
Alzheimer’s disease (AD) is a globally prevalent form of dementia that impacts diverse populations and is characterized by progressive neurodegeneration and impairments in executive memory. Although the exact mechanisms underlying AD pathogenesis remain unclear, it is commonly accepted that the aggregation of misfolded proteins, such as amyloid plaques and neurofibrillary tau tangles, plays a critical role. Additionally, AD is a multifactorial condition influenced by various genetic factors and can manifest as either early-onset AD (EOAD) or late-onset AD (LOAD), each associated with specific gene variants. One gene of particular interest in both EOAD and LOAD is RIN3, a guanine nucleotide exchange factor. This gene plays a multifaceted role in AD pathogenesis. Firstly, upregulation of RIN3 can result in endosomal enlargement and dysfunction, thereby facilitating the accumulation of beta-amyloid (Aβ) peptides in the brain. Secondly, RIN3 has been shown to impact the PICLAM pathway, affecting transcytosis across the blood-brain barrier. Lastly, RIN3 has implications for immune-mediated responses, notably through its influence on the PTK2B gene. This review aims to provide a concise overview of AD and delve into the role of the RIN3 gene in its pathogenesis.
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP and AMPA receptor (AMPAR) insertion during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes, and decreased AMPAR insertion during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and disinhibition of Rab10 signaling gates the induction of LTP and associated spine structural plasticity.
Macropinocytosis is the process in which active movements of the cell surface internalize extracellular fluid and solutes into 0.2–10 µm diameter endocytic vesicles called macropinosomes. Macropinocytosis is among the most conserved mechanisms of endocytosis and is present in protozoan and animal cells. It has been implicated in cellular feeding, antigen processing, cell motility and infection by viruses and bacteria. Macropinosomes form by localized movements of membrane and the actin cytoskeleton, leading to formation of cell surface ruffles, which close into macropinosomes. Nascent macropinosomes shrink after forming, the internalized membrane recycles to the cell surface, and ingested macromolecular solutes are degraded by acid hydrolases following delivery into lysosomes.
The RAS family of small GTPases are among the most frequently mutated oncogenes in human cancer. Approximately 20% of cancers harbor a RAS mutation, and > 150 different missense mutations have been detected. Many of these mutations have mutant-specific biochemical defects that alter nucleotide binding and hydrolysis, effector interactions and cell signaling, prompting renewed efforts in the development of anti-RAS therapies, including the mutation-specific strategies. Previously viewed as undruggable, the recent FDA approval of a KRASG12C-selective inhibitor has offered real promise to the development of allele-specific RAS therapies. A broader understanding of the mutational consequences on RAS function must be developed to exploit additional allele-specific vulnerabilities. Approximately 94% of RAS mutations occur at one of three mutational “hot spots” at Gly¹², Gly¹³ and Gln⁶¹. Further, the single-nucleotide substitutions represent > 99% of these mutations. Within this scope, we discuss the mutational frequencies of RAS isoforms in cancer, mutant-specific effector interactions and biochemical properties. By limiting our analysis to this mutational subset, we simplify the analysis while only excluding a small percentage of total mutations. Combined, these data suggest that the presence or absence of select RAS mutations in human cancers can be linked to their biochemical properties. Continuing to examine the biochemical differences in each RAS-mutant protein will continue to provide additional breakthroughs in allele-specific therapeutic strategies.
Epidermal growth factor (EGF)-receptor regulates several downstream signaling pathways upon EGF stimulation that involves cell proliferation, migration and invasion. Internalized EGF-receptor is either recycled or degraded, which fate is regulated in part by Ras interference 1 (RIN1). In this study, we tested the hypothesis that RIN1, a Ras effector protein and Rab5 guanine nucleotide exchange factor, controls several signaling molecules leading to the modulation of the telomerase activity; thus, allowing proper cell proliferation. We report that expression of RIN1 completely blocked proliferation of MCF-12 A and MCF-7 cells, while partially inhibited proliferation of MDA-MB-231 cells upon EGF stimulation. Furthermore, expression of the C-terminal region of RIN1 selectively plays a critical role in the inhibition of the proliferation of MDA-MB-231 cells. However, this inhibitory effect was specifically affected by the independent expression of RIN1:Vsp9 and RIN1:RA domains. Additionally, endogenous level of expression of RIN1 was decreased in metastatic MDA-MB-231 cells as compared with non-tumorigenic MCF-12 A cells. We observed that expression of RIN1:R94A mutant blocked the proliferation of MDA-MB-231 cells, while expression of RIN1:Y561F and RIN1:R629A mutants completely reversed the inhibitory effect of RIN1:WT. Consistent with our observations, we found that expression of RIN1:WT in MDA-MB-231 cells diminished both protein kinase B (AKT) and extracellular-signal-regulated kinase 1/2 (ERK1/2) activities while p38 mitogen-activated protein kinases (p38 MAPK) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) were unaffected, but it produced downregulation of cellular-myelocytomatosis (c-Myc), erythroblast transformation specific (Ets2) and signal transducer and activator of transcription 3 (Stat3) activities. Inversely, expression of high-mobility group box 1 (HMBG1) was inhibited whereas expression of forkhead box transcription factor 1 (FOXO1) was increased in cells expressing RIN1. Interestingly, expression of RIN1 blocked telomerase activity and human telomerase reverse transcriptase (hTERT) expression, which correlated with the downregulations of c-Myc, Ets-2 and Stat3 activation. Taken together these findings indicate that RIN1 is a critical player in the modulation of the telomerase activity as well as hTERT expression in MDA-MB-231 cells upon EGF stimulation.
The core machinery for vesicular membrane trafficking broadly comprises of coat proteins, RABs, tethering complexes and SNAREs. As cellular membrane traffic modulates key processes of mitogenic signaling, cell migration, cell death and autophagy, its dysregulation could potentially results in increased cell proliferation and survival, or enhanced migration and invasion. Changes in the levels of some components of the core machinery of vesicular membrane trafficking, likely due to gene amplifications and/or alterations in epigenetic factors (such as DNA methylation and micro RNA) have been extensively associated with human cancers. Here, we provide an overview of association of membrane trafficking with cancer, with a focus on mutations and variants of coat proteins, RABs, tethering complex components and SNAREs that have been uncovered in human cancer cells/tissues. The major cellular and molecular cancer-driving or suppression mechanisms associated with these components of the core membrane trafficking machinery shall be discussed.
Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein–protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes.
Significance
We establish a deep network of protein–protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.
This article is highlighted in the In This Issue feature, p. 1775
A key protein required for incorporating substances into cells has been identified by researchers in South Korea. During this process, called endocytosis, materials are engulfed by the cell membrane, then packaged into vesicles by organelles called endosomes for transport around the cell. The small enzyme Rab5 is involved in fusing and transporting vesicles through interactions with a protein called Gapex5. It has been unclear how these interactions occur, but Woo Keun Song and Yun Hyun Huh at Gwangju Institute of Science and Technology and co-workers suspected a role for SPIN90, an ‘adapter protein’ that helps connect other molecules. They found that Rab5 and Gapex5 did not come close in mutant cells lacking SPIN90. In normal cells, SPIN90 interacted strongly with both Rab5 and Gapex5, recruiting them to endosomes where they operated together on vesicle formation.
Growth factor receptors play a variety of roles during embryonic development and in adult homeostasis. These receptors are activated repeatedly in different cellular contexts and with different cellular outcomes. This begs the question as to how cells in a particular developmental, spatial and temporal context, or in adult tissue, interpret signalling by growth factor receptors in order to deliver qualitatively different signalling outputs. One mechanism by which this could occur is via endocytic regulation. The original paradigm for the role of endocytosis in growth factor receptor signalling was that receptor uptake has a quantitative role in signalling by reducing the number of cell surface receptors available for activation and targeting activated receptors for degradation. However, a range of studies over the last several years, in many different experimental systems, has demonstrated an additional qualitative role for endocytic trafficking in receptor signalling, with specific outcomes depending on the location of the signalling complex. Confinement of receptors within endosomes can spatially regulate signalling, facilitating specific protein interactions or post-translational modifications that alter throughout the trafficking process. Therefore, endocytosis does not simply regulate cell surface expression, but tightly controls protein interactions and function to produce distinct outcomes.
Endocytosis is a crucial process in eukaryotic cells. The GTPases Rab 5, 21 and 22 that mediate endocytosis are ancient eukaryotic features and all available evidence suggests conserved function. In animals and fungi, these GTPases are regulated in part by proteins possessing Vps9 domains. However, the diversity, evolution and functions of Vps9 proteins beyond animals or fungi are poorly explored. Here we report a comprehensive analysis of the Vps9 family of GTPase regulators, combining molecular evolutionary data with functional characterization in the non‐opisthokont model organism Trypanosoma brucei. At least three subfamilies, Alsin, Varp and Rabex5+GAPVD1, are found across eukaryotes, suggesting that all are ancient features of regulation of endocytic Rab protein function. There are examples of lineage‐specific Vps9 subfamily member expansions and novel domain combinations, suggesting diversity in precise regulatory mechanisms between individual lineages. Characterization of the Rabex5+GAPVD1 and Alsin orthologues in T. brucei demonstrates that both proteins are involved in endocytosis, and that simultaneous knockdown prevents membrane recruitment of Rab5 and Rab21, indicating conservation of function. These data demonstrate that, for the Vps9‐domain family at least, modulation of Rab function is mediated by evolutionary conserved protein‐protein interactions.
We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.
We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.
Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.
Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.
Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.
Insulin and epidermal growth factor receptors transmit signals for cell proliferation and gene regulation through formation of active GTP-bound p21ras mediated by the guanine nucleotide exchange factor Sos. Sos is constitutively bound to the adaptor protein Grb2 and growth factor stimulation induces association of the Grb2/Sos complex with Shc and movement of Sos to the plasma membrane location of p21ras. Insulin or epidermal growth factor stimulation induces a rapid increase in p21ras levels, but after several minutes levels decline toward basal despite ongoing hormone stimulation. Here we show that deactivation of p21ras correlates closely with phosphorylation of Sos and dissociation of Sos from Grb2, and that inhibition of mitogen-activated protein (MAP) kinase kinase (also known as extracellular signal-related kinase (ERK) kinase, or MEK) blocks both events, resulting in prolonged p21ras activation. These data suggest that a negative feedback loop exists whereby activation of the Raf/MEK/MAP kinase cascade by p21ras causes Sos phosphorylation and, therefore, Sos/Grb2 dissociation, limiting the duration of p21ras activation by growth factors. A serine/threonine kinase downstream of MEK (probably MAP kinase) mediates this desensitization feedback pathway.
Son of sevenless-1 and -2 (Sos-1 and -2) are guanosine nucleotide exchange factors implicated in the activation of Ras by both the insulin and epidermal growth factor signal transduction pathways. Ras appears to function by initiating the activation of cellular protein kinases including mitogen-activated protein (MAP) kinases. Sos proteins contain numerous sequences in their carboxyl-terminal regions which correspond to consensus sites for MAP kinase phosphorylation. To examine whether these sites are substrates for MAP kinases, the cDNA encoding Drosophila Sos (dSos) was tagged with sequences encoding the major antigenic epitope of the influenza virus hemagglutinin (HA) to create a dSosHA fusion construct. dSosHA was transiently expressed in COS-1 cells and immunoprecipitated with anti-HA antibodies. When immune complexes were incubated with purified MAP kinase and [gamma-32P]ATP, a phosphorylated band of 180 kDa was observed when analyzed by SDS-polyacrylamide gel electrophoresis. This band was not present in immunoprecipitations from cells transfected with vector alone. No phosphorylation of the 180 kDa band was seen when immunoprecipitates were incubated with [gamma-32P]ATP in the absence of MAP kinase. Two dimensional analysis of tryptic peptides from dSosHA phosphorylated by MAP kinase in vitro revealed two major phosphorylated species that were also found in dSosHA isolated from COS-1 cells labeled with 32Pi. These results are consistent with the hypothesis that a feedback loop exists wherein growth factor-activated MAP kinases phosphorylate and regulate Sos proteins.
Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.
Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2- SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.
Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated
endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling
events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for
full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling
but also in establishing and controlling specific signaling pathways.
Endocytosis in eukaryotic cells is characterized by the continuous and regulated formation of prolific numbers of membrane vesicles at the plasma membrane. These vesicles come in several different varieties, ranging from the actin-dependent formation of phagosomes involved in particle uptake, to smaller clathrin-coated vesicles responsible for the internalization of extracellular fluid and receptor-bound ligands. In general, each of these vesicle types results in the delivery of their contents to lysosomes for degradation. The membrane components of endocytic vesicles, on the other hand, are subject to a series of highly complex and iterative molecular sorting events resulting in their targeting to specific destinations. In recent years, much has been learned about the function of the endocytic pathway and the mechanisms responsible for the molecular sorting of proteins and lipids. This review attempts to integrate these new concepts with long-established views of endocytosis to present a more coherent picture of how the endocytic pathway is organized and how the intracellular transport of internalized membrane components is controlled. Of particular importance are emerging concepts concerning the protein-based signals responsible for molecular sorting and the cytosolic complexes responsible for the decoding of these signals.
Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates
that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling
via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus
vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression
of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated
pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant
of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis
but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent
membrane ruffling.
Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now
show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required
for efficient RAS binding. RIN1 interacts with the “effector domain” of RAS and employs some RAS determinants that are common
to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1
that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed
in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal
domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence
shows preferential binding to the ABL–SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition,
RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is
able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1
regulation.
Transient expression of oncogenic Ha-Ras (Ras:V12) stimulates endocytosis. Using NIH3T3 cells expressing constitutively active protein kinase B/akt (PKB/akt) or kinase-dead PKB/akt, we show that PKB/akt mediates the stimulatory effect of Ras on endocytosis. Fluid phase endocytosis of horseradish peroxidase in cells expressing the constitutively active form of PKB/akt was elevated and insensitive to phosphatidylinositol 3-kinase inhibitors. However, expression of dominant negative Rab5:N34 blocked endocytosis in cells expressing the constitutively active form of PKB/akt.
Transient expression of either Rab5:wt or Rab5:L79, a GTPase deficient mutant of Rab5, in cells expressing constitutively activated PKB/akt further increased endocytic rate. However, in cells expressing kinase-dead PKB/akt, endocytic rate was not affected by transient expression of Rab5:wt. Rab5:L79, on the other hand, increased endocytosis in cells expressing kinase-dead PKB/akt. Similar results were obtained using an in vitro endosome fusion reconstitution assay with cytosol prepared from cells expressing the activated PKB/akt or kinase-dead PKB/akt. Both Rab5:wt and Rab5:L79 stimulated endosome fusion when assayed in cytosol containing the activated PKB/akt, whereas only Rab5:L79 activated fusion when the assay utilized cytosol from kinase-dead expressing cells. We conclude that Ras activation of endocytosis requires both PKB/akt and Rab5 and that active kinase is required for activation Rab5.
Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.
Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.
Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants,VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein
transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association
as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or
GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which
is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport.
Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins.
We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no
obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein
transport.
Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.
Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc.Grb2.Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-alpha, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.
Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.
Son of sevenless-1 and -2 (Sos-1 and -2) are guanosine nucleotide exchange factors implicated in the activation of Pas by both the insulin and epidermal growth factor signal transduction pathways. Pas appears to function by initiating the activation of cellular protein kinases including mitogen-activated protein (MAP) kinases. Sos proteins contain numerous sequences in their carboxylterminal regions which correspond to consensus sites for MAP kinase phosphorylation. To examine whether these sites are substrates for MAP kinases, the cDNA encoding Drosophila Sos (dSos) was tagged with sequences encoding the major antigenic epitope of the influenza virus hemagglutinin (HA) to create a dSosHA fusion construct. dSosHA was transiently expressed in COS-1 cells and immunoprecipitated with anti-HA antibodies. When immune complexes were incubated with purified MAP kinase and [gamma-P-32]ATP, a phosphorylated band of 180 kDa was observed when analyzed by SDS-polyacrylamide gel electrophoresis. This band was not present in immunoprecipitations from cells transfected with vector alone. No phosphorylation of the 180 kDa band was seen when immunoprecipitates were incubated with [gamma(32)P]ATP in the absence of MAP kinase. Two dimensional analysis of tryptic peptides from dSosHA phosphorylated by MAP kinase in vitro revealed two major phosphorylated species that were also found in dSosHA isolated from COS-1 cells labeled with P-32(i). These results are consistent with the hypothesis that a feedback loop exits wherein growth factor-activated MAP kinases phosphorylate and regulate Sos proteins.
CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Timelapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a 'bridge' fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous 'bridge' between them. The unique aspect of rab5 mediated 'bridge' fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion 'bridge', we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFPrab5 into a brightly fluorescent spot in the 'bridge' region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion 'bridges'. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFPrab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (0.3) xlO-9cm2/second.
We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.
The EGF receptor (EGFR) upon activation signals increased cell movement. However, the domains within the receptor, and the pathway which trigger movement are undefined. We expressed EGFR mutants at physiologic levels in receptor-devoid NR6 cells to investigate this biologic response. The receptors possessed kinase activity and underwent autophosphorylation as predicted by primary amino acid sequence. EGF-induced cell motility was assessed in vitro by excess migration into an acellular area and colony scatter in the presence of saturating concentrations of EGF. Wild-type (WT)-EGFR signaled increased motility. However, replacing the conserved lysine721 with methionine resulted in a kinase-inactive receptor which did not elicit movement. Removal of the entire terminus by truncation (c'973) also abrogated ligand-induced motility. Thus, we concentrated on the carboxy-terminal domains. EGF-induced movement was seen with a less-truncated mutant (c'1000) that contained a single autophosphorylated tyrosine (tyrosine992). Other mutants, c'991 and c'1000F992, in which this tyrosine was removed did not signal motility. Fusion mutants which presented other autophosphorylated tyrosine domains also exhibited EGF-induced movement. These findings suggested that the presence of both an autophosphorylated tyrosine signaling domain and the kinase activity are necessary for this biologic response. All kinase-positive mutants signaled cell proliferation but only those that contained autophosphorylatable tyrosines induced movement. The motility responses mediated by these EGFR were identical in the presence or absence of mitomycin-C, at a dose (0.5 micrograms/ml) which completely inhibited cell proliferation. On the other side, D-actinomycin (50 ng/ml) blocked EGF-induced motility but did not affect thymidine incorporation. Thus, EGF-induced mitogenesis and cell motility are mediated through different pathways.
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We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5ile 133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a ratelimiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.
Recent years have witnessed tremendous growth in the epidermal growth factor (EGF) family of peptide growth factors and the ErbB family of tyrosine kinases, the receptors for these factors. Accompanying this growth has been an increased appreciation for the roles these molecules play in tumorigenesis and in regulating cell proliferation and differentiation during development. Consequently, a significant question has been how diverse biological responses are specified by these hormones and receptors. Here we discuss several characteristics of hormone-receptor interactions and receptor coupling that contribute to specificity: 1) a single EGF family hormone can bind multiple receptors; 2) a single ErbB family receptor can bind multiple hormones; 3) there are three distinct functional groups of EGF family hormones; 4) EGF family hormones can activate receptors in trans, and this heterodimerization diversifies biological responses; 5) ErbB3 requires a receptor partner for signaling; and 6) ErbB family receptors differentially couple to signaling pathways and biological responses. BioEssays 20:41–48, 1998. © 1998 John Wiley & Sons, Inc.
Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.
Receptor oligomerization was initially proposed as a mechanism by which epidermal growth factor activates the protein tyrosine kinase activity of its receptor. It is now well established that ligand-induced receptor oligomerization plays an important role in transmembrane signaling by a large number of receptors for hormones, cytokines and growth factors. Heterodimerization of the extracellular domains of two members of the same receptor family, or interaction with an accessory molecule, can increase the diversity of ligands recognized by individual receptors. Heterodimerization of cytoplasmic domains permits the recruitment of different complements of SH2-domain-containing signaling molecules, increasing the repertoire of signaling pathways that can be activated by a given receptor.
The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.
We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.
Identification of a mutant epidermal growth factor (EGF) receptor that does not undergo downregulation has provided a genetic probe to investigate the role of internalization in ligand-induced mitogenesis. Contact-inhibited cells expressing this internalization-defective receptor exhibited a normal mitogenic response at significantly lower ligand concentrations than did cells expressing wild-type receptors. A transformed phenotype and anchorage-independent growth were observed at ligand concentrations that failed to elicit these responses in cells expressing wild-type receptors. These findings imply that activation of the protein tyrosine kinase activity at the cell membrane is sufficient for the growth-enhancing effects of EGF. Thus, downregulation can serve as an attenuation mechanism, without which transformation ensues.
Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.
We have located the distal boundary of the tyrosine kinase domain of the EGF receptor and have identified a distinct sequence in the C' terminus required for EGF-dependent receptor internalization, leading to receptor down-regulation and degradation. Within this receptor domain, an 18 amino acid highly negatively charged region of predicted helical structure is required both for endocytosis via a high-affinity, saturable pathway and for ligand-stimulated increases in cytosolic calcium. In contrast to kinase-inactive, internalization-competent receptors, kinase-active, internalization-defective receptors effectively signaled gene transcription, morphological transformation, and growth. These observations support the hypothesis that mitogenic responses to EGF are mediated by activation of the intrinsic protein tyrosine kinase activity of the membrane-bound receptor, with ligand-induced internalization serving to terminate the signal.
Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.
The mouse protein mSos1 has a central Ras guanine nucleotide exchange domain, and a long proline-rich C-terminal tail which contains several potential binding sites for the SH3 domains of the adaptor protein, Grb2. In fibroblasts, growth factor stimulation results in the recruitment of Grb2-mSos1 into complexes with activated receptors and cytoplasmic phosphoproteins such as Shc, which are apparently involved in Ras activation, and subsequently to an increase in mSos1 phosphorylation on serine and threonine. The catalytic and C-terminal domains of mSos1 contain several potential sites for phosphorylation by mitogen-activated protein kinases. In vitro, purified p42/p44 MAP-kinase selectively phosphorylated the C-terminal tail of mSos1. Comparative tryptic phosphopeptide mapping of mSos1 phosphorylated in vitro by MAP kinase and of mSos1 immunoprecipitated from EGF-stimulated cells, revealed several phosphopeptides in common. These common phosphorylation sites have been mapped to a region encompassing the first three proline (pro)-rich motifs in the tail of mSos1. Furthermore, a region of mSos1 containing the first two pro-rich motifs could associate with MBP kinase activity in vitro. Phosphorylation of mSos1 did not affect binding of Grb2 to mSos1, but appeared to decrease binding of the mSos1-Grb2 complex to Shc and the EGF-receptor. These findings suggest a potential inhibitory role for MAP-kinase in attenuating nucleotide exchange on Ras, by uncoupling mSos1 from membrane-bound receptor complexes that lead to Ras activation.
The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces
cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating
mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein
encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named
this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP.
Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a
human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of
Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides
primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells
as an effector or regulator of H-Ras.
We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.
Association of the p21ras guanine nucleotide exchange factor mSOS with tyrosine-phosphorylated Shc has been implicated in the activation of p21ras. In addition, after growth factor stimulation mSOS becomes phosphorylated as indicated by the appearance of a form of mSOS with reduced electrophoretic mobility. This phosphorylation is delayed with respect to Shc-Grb2-mSOS complex formation and activation of p21ras. To investigate the role of mSOS phosphorylation in further detail we have investigated the effect of phosphorylation on mSOS complex formation and p21ras activation. We found that Shc is associated with the unphosphorylated, faster migrating form of mSOS. Furthermore, although there is a correlation between the amount of complexes formed and the activation of p21ras, there is no such a correlation between mSOS phosphorylation and p21ras activation. In addition, inhibition of mSOS phosphorylation did not affect complex formation of mSOS with tyrosine phosphorylated Shc. Also, induction of mSOS phosphorylation prior to complex formation did not affect EGF-induced association of mSOS with Shc significantly, and Shc still associated predominantly with the faster migrating form of mSOS. From these results we conclude that the unphosphorylated form of mSOS is associated with Shc and that perhaps a phosphorylation-dephosphorylation step is part of the mSOS activation-inactivation cycle.
A two-hybrid protein interaction screen was used to isolate cDNAs encoding human proteins that can interact with human CDK2 in yeast. A new member of the retinoblastoma susceptibility gene family, Rbr-2 (Rb-related), was obtained. The sequence of the Rbr-2 protein shares approximately 50% identify with p107 and homology to Rb within the pocket domain. Several lines of evidence indicate that Rbr-2 is the adenovirus E1A-associated p130. Like Rb and p107, p130Rbr-2 can bind to viral oncoproteins, SV40 large T antigen, and adenovirus E1A through its pocket domain. Although p130Rbr-2 does not bind to CDK2 in vitro, it can interact with cyclins, with a clear preference for D-type cyclins. Because both CDK2 and p130Rbr-2 show affinity for cyclins, we suggest that p130Rbr-2 and CDK2 interacted through a yeast-derived cyclin bridge in the two-hybrid screen. The gene encoding p130Rbr-2 mapped to 16q13, a region of frequent genomic alteration in human tumors.
We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.
In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 was phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resulted in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylated hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 was phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping showed that MAPK phosphorylated hSOS1 in vitro on sites which were also phosphorylated in vivo. Phosphorylation by MAPK did not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex showed that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 was unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway.
Pinocytosis and membrane ruffling are among the earliest and most dramatic cellular responses to stimulation by growth factors or other mitogens. The small Ras-related G proteins Rho and Rac have a regulatory role in membrane ruffling and activated Rho has been shown to stimulate pinocytosis when microinjected into Xenopus oocytes. In contrast to these well established effects of Rho and Rac on plasma membrane morphology and bulk pinocytosis, there has been no evidence for their involvement in the regulation of receptor-mediated endocytosis in clathrin-coated pits. Here we show that activated Rho and Rac inhibit transferrin-receptor-mediated endocytosis when expressed in intact cells. Furthermore, we have reconstituted these effects in a cell-free system and established that Rho and Rac can regulate clathrin-coated vesicle formation.
RIN1 was originally identified by its ability to physically bind to and interfere with activated Ras in yeast. Paradoxically, RIN1 potentiates the oncogenic activity of the BCR-ABL tyrosine kinase in hematopoietic cells and dramatically accelerates BCR-ABL-induced leukemias in mice. RIN1 rescues BCR-ABL mutants for transformation in a manner distinguishable from the cell cycle regulators c-Myc and cyclin D1 and the Ras connector Shc. These biological effects require tyrosine phosphorylation of RIN1 and binding of RIN1 to the Abl-SH2 and SH3 domains. RIN1 is tyrosine phosphorylated and is associated with BCR-ABL in human and murine leukemic cells. RIN1 exemplifies a new class of effector molecules dependent on the concerted action of the SH3, SH2, and catalytic domains of a cytoplasmic tyrosine kinase.
The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.
The receptor for the epidermal growth factor (EGF) and related ligands (EGFR), the prototypal member of the superfamily of receptors with intrinsic tyrosine kinase activity, is widely expressed on many cell types, including epithelial and mesenchymal lineages. Upon activation by at least five genetically distinct ligands (including EGF, transforming growth factor-alpha (TGF alpha) and heparin-binding EGF (HB-EGF)), the intrinsic kinase is activated and EGFR tyrosyl-phosphorylates itself and numerous intermediary effector molecules, including closely-related c-erbB receptor family members. This initiates myriad signaling pathways, some of which attenuate receptor signaling. The integrated biological responses to EGFR signaling are pleiotropic including mitogenesis or apoptosis, enhanced cell motility, protein secretion, and differentiation or dedifferentiation. In addition to being implicated in organ morphogenesis, maintenance and repair, upregulated EGFR signaling has been correlated in a wide variety of tumors with progression to invasion and metastasis. Thus, EGFR and its downstream signaling molecules' are targets for therapeutic interventions in wound repair and cancer.