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SPAR2, a novel SPAR-related protein with GAP activity for Rap1 and Rap2
Journal of Neurochemistry
Abstract
Spine-associated RapGAP 2 (SPAR2) is a novel GTPase activating protein (GAP) for the small GTPase Rap that shows significant sequence homology to SPAR, a synaptic RapGAP that was reported to regulate spine morphology in hippocampal neurons. SPAR2, like SPAR, interacts with the recently described synaptic scaffolding protein ProSAP-interacting protein (ProSAPiP), which in turn binds to the PDZ domain of ProSAP/Shank post-synaptic density proteins. In subcellular fractionation experiments, SPAR2 is enriched in synaptosomes and post-synaptic density fractions indicating that it is a synaptic protein. Furthermore, we could show using in vitro GAP assays that SPAR2 has GAP activity for Rap1 and Rap2. Expression in COS-7 cells, however, revealed different actin-binding properties of SPAR2 and SPAR. Additionally, over-expression of SPAR2 in cultured hippocampal neurons did not affect spine morphology as it was reported for SPAR. In situ hybridization studies also revealed a differential tissue distribution of SPAR and SPAR2 with SPAR2 transcripts being mainly expressed in cerebellar and hippocampal granule cells. Moreover, in the cerebellum SPAR2 is developmentally regulated with a peak of expression around the period of synapse formation. Our results imply that SPAR2 is a new RapGAP with specific functions in cerebellar and hippocampal granule cells.
... Sipa1l1/SPAR is localized within postsynaptic specializations of spine synapses, attached to actin filaments via two actin-binding domains and widely expressed in the forebrain throughout postnatal development (Pak et al. 2001;Roy et al. 2002;Wendholt et al. 2006;Spilker et al. 2008;Schmeisser et al. 2009). Furthermore, Sipa1l1/SPAR is essential for shaping the morphology of dendritic spines and synaptic growth and strength can both be regulated via tight control of Sipa1l1/SPAR expression levels at the synapse (Pak and Sheng 2003;Maruoka et al. 2005;Ang et al. 2008;Seeburg et al. 2008;Hoe et al. 2009;Lu et al. 2009;Herrick et al. 2010;Chen et al. 2012;Mihalas et al. 2013). ...
... Sipa1l2/SPAR2 is also localized within postsynaptic specializations, but not attached to actin filaments and has no effect on dendritic spine shape. Interestingly, its expression pattern in brain is quite restricted as Sipal12/ SPAR2 transcripts are predominantly found in granule cells of the cerebellum and the dentate gyrus (Spilker et al. 2008). ...
... The pEGFP-SPAR3 constructs for over-expression of truncated Sipa1l3/SPAR3 variants (SPAR3DC-term, SPAR3C-term, SPAR3C-term-DCC, SPAR3C-term-CC) were cloned in a PCR-based approach using the full-length Sipa1l3/SPAR3 cDNA sequence as a template. The following constructs have been described elsewhere: pEGFP-SPAR, pEGFP-SPAR2 (both Spilker et al. 2008) and pDsRed-ProSAPiP1 (Wendholt et al. 2006). ...
Rap GTP ase‐activating proteins (Rap GAP s) are essential for synaptic function as they tightly regulate synaptic Rap signaling. Among the most abundant synaptic Rap GAP s in brain are the Spine‐associated Rap GAP s ( SPAR s) Sipa1l1/ SPAR and Sipa1l2/ SPAR 2, whereas nothing has been reported on Sipa1l3/ SPAR 3. In this study, we show that Sipa1l3/ SPAR 3 is conserved across species, has a distinct expression pattern in the developing rat brain and is localized at excitatory postsynapses. We further demonstrate that the Sipa1l3/ SPAR 3 C‐terminus is required for postsynaptic targeting and represents an interaction module for Fezzins such as Pro SAP iP1/Lzts3, a binding partner of the postsynaptic scaffold protein Shank3. Taken together, our data imply that Sipa1l3/ SPAR 3 is a hitherto unknown synaptic Rap GAP , which is targeted to postsynaptic specializations and interacts with Fezzins.
image Spine‐associated RapGAPs (SPARs) are essential modulators of synaptic signaling. Our study is the first to characterize the SPAR family member Sipa1l3/SPAR3 in neuronal tissue. We show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in brain and is localized to excitatory postsynapses via its C‐terminus, which represents an interaction module for other postsynaptic proteins including the Fezzin ProSAPiP1/Lzts3.
... SIPA1L2 (also known as SPAR2) is a member of the SIPA1L family of neuronal RapGAPs ( Fig. S1) (11). The protein is most abundant in granule cells of the dentate gyrus (DG) and cerebellum and shows RapGAP activity for the small GTPases Rap1 and 2 (12). Here, we report that SIPA1L2 binds directly to TrkB, and links the receptor tyrosine kinase to a Dynein motor for retrograde trafficking via a direct interaction with the adaptor protein Snapin. ...
... doi: bioRxiv preprint first posted online Feb. 20, 2019; b. Scheme showing the domain organization and interaction motifs of SIPA1L2 with TrkB, Snapin, LC3 and ProSAPiP1 (12). (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Amphisomes are transient organelles that derive from fusion of autophagosomes with late endosomes. They rapidly transform into degradative autolysosomes, whereas non-degradative roles of the autophagic pathway have been barely described. Here we show that in neurons BDNF/TrkB receptor bearing Rab7 / Light chain 3 (LC3) - positive amphisomes signal at presynaptic boutons during retrograde trafficking to the soma. Local signaling and inward transport essentially require the Rap GTPase-activating (RapGAP) protein SIPA1L2, which directly binds to TrkB and Snapin to connect TrkB-containing amphisomes to dynein. Association with LC3 regulates the RapGAP activity of SIPA1L2 and thereby retrograde trafficking. Following induction of presynaptic plasticity amphisomes dissociate from dynein at boutons, and this enables local signaling and promotes transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Collectively, the data suggest that TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.
... We identified 4 strongly associated intronic variants in signal-induced proliferation-associated 1 like 2 (SIPA1L2), also known as spineassociated RapGAP2 (SPAR2), a member of the Rap GTPase-activating proteins (RapGAPs) enriched at synaptic sites. [12][13][14] Our functional studies imply a role in peripheral nerve myelination and regulating PMP22 expression. Combined with the genomic evidence, these results support SIPA1L2 as a potential modifier gene for CMT1A. ...
... 31,32 Studies suggest SIPA1L2 also has GTPase activating protein activity and functions in cerebellar and hippocampal granule cells. 13 In previous GWAS studies, SIPA1L2 was identified as a risk locus (lead SNP rs10797576, chr1:232,664,611) for Parkinson disease in European 43 and Iranian populations. 44 Importantly, our functional experiments support a role for SIPA1L2 in Schwann cell biology. ...
Objective
Genetic modifiers in rare disease have long been suspected to contribute to the considerable variance in disease expression, including Charcot‐Marie‐Tooth disease type 1A (CMT1A). To address this question the Inherited Neuropathy Consortium collected a large standardized sample of such rare CMT1A patients over a period of eight years. CMT1A is caused in most patients by a uniformly sized 1.5Mb duplication event involving the gene PMP22.
Methods
We genotyped DNA samples from 971 CMT1A patients on Illumina beadchips. Genome‐wide analysis was performed in a subset of 330 of these patients, who expressed the extremes of a hallmark symptom: mild and severe foot dorsiflexion strength impairment. SIPA1L2 (signal induced proliferation associated 1 like 2), the top identified candidate modifier gene, was expressed in the peripheral nerve and our functional studies identified and confirmed interacting proteins using co‐immunoprecipitation analysis, mass spectrometry, and immunocytochemistry. Chromatin immunoprecipitation and in vitro siRNA experiments were used to analyze gene regulation.
Results
We identified significant association of four SNPs (rs10910527, rs7536385, rs4649265, rs1547740) in SIPA1L2 with foot dorsiflexion strength (P < 1×10‐7). Co‐immunoprecipitation and mass‐spectroscopy studies identified β‐actin and MYH9 as SIPA1L2 binding partners. Further, we show that SIPA1L2 is part of a myelination‐associated co‐expressed network regulated by the master transcription factor SOX10. Importantly, in vitro knock‐down of SIPA1L2 in Schwannoma cells lead to a significant reduction of PMP22 expression, hinting at a potential strategy for drug development.
Interpretation
offers a new pathway to therapeutic interventions.
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... They further exhibit coiled-coil domains mediating homo-and heteromultimerization among family members and bind to Spine-associated Rap GTPaseactivating proteins (SPARs), essential modulators of spine morphology. It is thus hypothesized that Fezzins contribute to synaptic function by interconnecting Shanks and SPARs at the PSD (Maruoka et al., 2005;Wendholt et al., 2006;Spilker et al., 2008;Schmeisser et al., 2009;Mayanagi et al., 2015;Dolnik et al., 2016). However, mechanistic data have only been obtained for PSD-Zip70, which was shown to be critical for mature spine formation and the maintenance of spine maturity involving both SPAR and Rap2 signaling (Maruoka et al., 2005;Mayanagi et al., 2015). ...
... Both the full length ProSAPiP1 cDNA sequence (Wendholt et al., 2006) and RNAi oligonucleotides purchased from Eurofins targeting exon 3 of ProSAPiP1 (5 -GCCTTCAAGCCTGTTGTAC-3 ) were cloned into the FUGW vector system. The GFP-SPAR2, GFP-SPAR3 and SPAR-RNAi constructs have been described elsewhere (Richter et al., 2007;Spilker et al., 2008;Dolnik et al., 2016). ...
The postsynaptic density or PSD is a submembranous compartment containing a wide array of proteins that contribute to both morphology and function of excitatory glutamatergic synapses. In this study, we have analyzed functional aspects of the Fezzin ProSAPiP1, an interaction partner of the well-known PSD proteins Shank3 and SPAR. Using lentiviral-mediated overexpression and knockdown of ProSAPiP1, we found that this protein is dispensable for the formation of both pre- and postsynaptic specializations per se. We further show that ProSAPiP1 regulates SPAR levels at the PSD and the maturation of dendritic spines. In line with previous findings on the ProSAPiP1 homologue PSD-Zip70, we conclude that Fezzins essentially contribute to the maturation of excitatory spine synapses.
... Sipa1 (signal-induced proliferation-associated 1, originally named Spa-1) and spineassociated RapGAP 1 [Spar1, also called Sipa1 like 1 (Sipa1l1)], Spar2, and -3 (Sipa1l2, -3) inactivate Rap1 and Rap2 (Pak et al., 2001;Roy et al., 2002;Spilker et al., 2008;. All contain a GAP, a PDZ and a C-terminal guanylate kinase (GUK)-binding domain (GKBD) N-and C-terminus (Act1 and Act2) were found in Spar1, which are absent in Sipa1. ...
... Epac1/2 double knockout mice show more severe deficits in synaptic plasticity and a reduced release of glutamate from presynaptic terminals (Yang et al., 2012). Spar1 interacts with scaffolding proteins in the PSD and is subject to multiple regulatory mechanisms (Pak et al., 2001;Roy et al., 2002;Spilker et al., 2008). The GKBD of Spar1 -3 binds to PSD-95 and Lzts1 (leucine zipper, putative tumor suppressor 1, also called PSD-Zip70), Lzts2 (Lapser1), Lzts3 (Prosapip1) and Lzts4 (N4BP3) (Dolnik et al., 2016;Maruoka et al., 2005;Schmeisser et al., 2009;Wendholt et al., 2006). ...
Small GTPases are central regulators of many cellular processes. The highly conserved Rap GTPases perform essential functions in the mammalian nervous system during development and in mature neurons. During neocortical development, Rap1 is required to regulate cadherin- and integrin-mediated adhesion. In the adult nervous system Rap1 and Rap2 regulate the maturation and plasticity of dendritic spine and synapses. Although genetic studies have revealed important roles of Rap GTPases in neurons, their regulation by guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them by stimulating their intrinsic GTPase activity is just beginning to be explored in vivo. Here we review how GEFs and GAPs regulate Rap GTPases in the nervous system with a focus on their in vivo function.
... In addition, SIPA1L1 harbors two regions of low homology found to associate with actin (19,20). Other members of the SIPA1L family include SIPA1L2 and SIPA1L3 (21,22). Expression of SIPA1L1 and SIPA1L2 (also called SPAR1 and 2) is enriched in hippocampal neurons, and functional roles have been identified in shaping dendritic spine morphology (19,22,23). ...
... Other members of the SIPA1L family include SIPA1L2 and SIPA1L3 (21,22). Expression of SIPA1L1 and SIPA1L2 (also called SPAR1 and 2) is enriched in hippocampal neurons, and functional roles have been identified in shaping dendritic spine morphology (19,22,23). To date, there is a lack of information about the functional role of SIPA1L3. ...
Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1 with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1, and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition (EMT) in the lens. Patient 2 with cataracts, was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.
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... Whereas Ras promotes LTP, Rap1 and Rap2 mediate LTD and depotentiation, respectively (Zhu et al. 2005). In contrast to the RasGAP SynGAP, a postsynaptic RapGAP SPAR, but not SPAR2, binds actin and promotes the growth of spines (Pak et al. 2001, Pak and Sheng 2003, Spilker et al. 2008. All these data suggest that Ras and Rap might act antagonistically in the postsynaptic compartment to regulate synapse strength and spine morphology. ...
The large majority of excitatory synapses are located on dendritic spines which are discrete membrane protrusions present on neuronal dendrites. Interestingly the highly heterogeneous morphology of dendritic spines is thought to be the morphological basis for synaptic plasticity associated to learning and memory formation. Indeed dendritic spines structure is regulated by molecular mechanisms that are fine tuned and adjusted according to level and direction of synaptic activity, development, specific brain region, and different experimental behavioral conditions. This supports the idea that reciprocal changes between the structure and function of spines impact both local and global integration of signals within dendrites. An increasing number of proteins have been found to be morphogens for dendritic spines and provided new insights into the molecular mechanisms regulating spine formation and morphology. Thus determining the mechanisms that regulate spine formation and morphology is essential for understanding the cellular changes that underlie learning and memory in normal and pathological conditions.
... A regulator of Rap1 is SIPA1L2 (also known as SPAR2), a member of the SIPA1L family of neuronal RapGAPs (Supplementary Fig. 1b-d) 9 . The protein is most abundant in granule cells of the dentate gyrus (DG) and cerebellum and shows RapGAP activity for Rap1 and 2 10 . This RapGAP activity promotes the intrinsic GTPase activity of Rap1/2 that catalyzes the hydrolysis of GTP to GDP and inactivates Rap1/2 and consequently, ERK signaling. ...
Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity. There is growing evidence that autophagy might serve specialized functions in neurons besides its role in protein homeostasis. In this study, authors demonstrate that axonal retrograde transport of BDNF/TrkB in neuronal amphisomes is involved in plasticity-relevant local signaling at presynaptic boutons and that SIPA1L2, a member of the SIPA1L family of neuronal RapGAPs, associates via LC3b to TrkB-containing amphisomes to regulate its motility and signaling at the axon terminals
... All Sipa family members share common domains, namely an N-terminal RapGAP domain (Rap-GTPase activating domain), a PDZ domain and a Cterminal coiled-coil domain that was found to harbor a leucine zipper (Wendholt et al., 2006). So far, Sipa1l1-3 have been analyzed mainly with respect to their synaptic function in the central nervous system (Dolnik et al., 2016;Pak et al., 2001;Spilker et al., 2008). ...
The signal-induced proliferation associated family of proteins comprises four members, SIPA1 and SIPA1L1-1L3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts up-stream of Sipa1l3 during eye development. Both, Sipa1l3 and Epha4 are required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrated that canonical Wnt signaling is inhibited downstream of Epha4/Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an up-regulation of axin2 expression, a direct Wnt/β-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/β-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells resulting in congenital cataracts.
... SPAR can interact with the PDZ domain of Shank using ProSAPiP1 as linker molecule [92]. It was first identified in a yeast two-hybrid screen using the guanylate kinase (GK) domain of the PSD-95 family member PSD-93 [93,94]. SPAR acts as a GAP protein for Rap small GTPases. ...
Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and í µí»½PIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.
... A major regulator of Ras activity in spines is the RasGAP protein SynGAP that plays a major inhibitory role by developmentally repressing excitability synapse and dendritic spine formation during the critical period (86,528). On the other hand, neurons overexpressing the postsynaptic RapGAPs SPAR (190,375,376,476) and Rap1GAP (313) have spines with altered morphology and larger head. Thus these results suggest that Ras and Rap, in the postsynaptic compartment, have an opposite effect in controlling synapse strength, spine morphology, and synaptic plasticity. ...
The introduction of high-resolution time lapse imaging and molecular biological tools has changed dramatically the rate of progress towards the understanding of the complex structure-function relations in synapses of central spiny neurons. Standing issues, including the sequence of molecular and structural processes leading to formation, morphological change, and longevity of dendritic spines, as well as the functions of dendritic spines in neurological/psychiatric diseases are being addressed in a growing number of recent studies. There are still unsettled issues with respect to spine formation and plasticity: Are spines formed first, followed by synapse formation, or are synapses formed first, followed by emergence of a spine? What are the immediate and long-lasting changes in spine properties following exposure to plasticity-producing stimulation? Is spine volume/shape indicative of its function? These and other issues are addressed in this review, which highlights the complexity of molecular pathways involved in regulation of spine structure and function, and which contributes to the understanding of central synaptic interactions in health and disease.
... 78 Ubiquitination can act as an important signal complex SCF (β-TrCP) leading to its degradation and regulation of actin polymerization and neuron growth. 91,92 Additionally, the Rac GAP α1-Chimaerin also undergoes polyubiquitination in neurons resulting in its degradation with resultant enhanced Rac activation. Although the precise roles of these pathways are unknown, the ubiquitination of these proteins may well play a key role in axon generation and neural signaling. ...
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
... Whereas Ras promotes LTP, Rap1 and Rap2 mediate LTD and depotentiation, respectively (Zhu et al. 2005). In contrast to the RasGAP SynGAP, a postsynaptic RapGAP SPAR, but not SPAR2, binds actin and promotes the growth of spines (Pak et al. 2001, Pak and Sheng 2003, Spilker et al. 2008. All these data suggest that Ras and Rap might act antagonistically in the postsynaptic compartment to regulate synapse strength and spine morphology. ...
The large majority of excitatory synapses are located on dendritic spines which are discrete membrane protrusions present on neuronal dendrites. Interestingly the highly heterogeneous morphology of dendritic spines is thought to be the morphological basis for synaptic plasticity associated to learning and memory formation. Indeed dendritic spines structure is regulated by molecular mechanisms that are fine tuned and adjusted according to level and direction of synaptic activity, development, specific brain region, and different experimental behavioral conditions. This supports the idea that reciprocal changes between the structure and function of spines impact both local and global integration of signals within dendrites. An increasing number of proteins have been found to be morphogens for dendritic spines and provided new insights into the molecular mechanisms regulating spine formation and morphology. Thus determining the mechanisms that regulate spine formation and morphology is essential for understanding the cellular changes that underlie learning and memory in normal and pathological conditions.
Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), leading to muscle weakness and loss of sensation in the hands and feet. A recent case-only genome-wide association study of CMT1A patients conducted by the Inherited Neuropathy Consortium identified a strong association between strength of foot dorsiflexion and variants in signal induced proliferation associated 1 like 2 (SIPA1L2), indicating that it may be a genetic modifier of disease. To validate SIPA1L2 as a candidate modifier and to assess its potential as a therapeutic target, we engineered mice with deletion of exon 1 (including the start codon) of the Sipa1l2 gene and crossed them to the C3-PMP22 mouse model of CMT1A. Neuromuscular phenotyping showed that Sipa1l2 deletion in C3-PMP22 mice preserved muscular endurance assayed by inverted wire hang duration and changed femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggest involvement of Sipa1l2 in cholesterol biosynthesis, a pathway that is also implicated in C3-PMP22 mice. Although Sipa1l2 deletion did impact CMT1A-associated phenotypes, thereby validating a genetic interaction, the overall effect on neuropathy was mild.
Despite numerous studies on various surface modifications on titanium and its alloys, it remains unclear what kind of titanium-based surface modifications are capable of controlling cell activity. This study aimed to understand the mechanism at the cellular and molecular levels and investigate the in vitro response of osteoblastic MC3T3-E1 cultured on the Ti-6Al-4V surface modified by plasma electrolytic oxidation (PEO) treatment. A Ti-6Al-4V surface was prepared by PEO at 180, 280, and 380 V for 3 or 10 min in an electrolyte containing Ca2+/Pi ions. Our results showed that PEO-treated Ti-6Al-4V-Ca2+/Pi surfaces enhanced the cell attachment and differentiation of MC3T3-E1 compared to the untreated Ti-6Al-4V control but did not affect cytotoxicity as shown by cell proliferation and cell death. Interestingly, on the Ti-6Al-4V-Ca2+/Pi surface treated by PEO at 280 V for 3 or 10 min, MC3T3-E1 showed a higher initial adhesion and mineralization. In addition, the alkaline phosphatase (ALP) activity significantly increased in MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi (280 V for 3 or 10 min). In RNA-seq analysis, the expression of dentin matrix protein 1 (DMP1), sortilin 1 (Sort1), signal-induced proliferation-associated 1 like 2 (SIPA1L2), and interferon-induced transmembrane protein 5 (IFITM5) was induced during the osteogenic differentiation of MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi. DMP1 and IFITM5 silencing decreased the expression of bone differentiation-related mRNAs and proteins and ALP activity in MC3T3-E1. These results suggest that the PEO-treated Ti-6Al-4V-Ca2+/Pi surface induces osteoblast differentiation by regulating the expression of DMP1 and IFITM5. Therefore, surface microstructure modification through PEO coatings with Ca2+/Pi ions could be used as a valuable method to improve biocompatibility properties of titanium alloys.
SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of GPCR signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1 -/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1 -/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.SIGNIFICANCE STATEMENTSIPA1L1 is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified GPCR-signaling regulators. Moreover, SIPA1L1 KO mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
Amyloid-β peptide (Aβ) is believed to be a primary cause of Alzheimer's disease. Many studies have demonstrated that Aβ causes morphological and functional alterations of dendritic spines, leading to synaptic dysfunction, but the effect of Aβ on damage to synaptic functions is not fully understood. Spine-associated Rap guanosine triphosphatase-activating protein (SPAR) is an important regulator of activity-dependent remodeling of synapses and is critically involved in both mature dendritic spine formation and the maintenance of spine maturity. Serum-inducible kinase (SNK) is an activity-inducible member of the polo-like family of serine/threonine kinases. Coordinated regulation of Ras and Rap by SNK is critical for homeostatic plasticity and memory. A previous study in which rats were injected with Aβ1–40 into the hippocampus showed that the SNK and SPAR signaling pathway may play a crucial role in Aβ-induced excitotoxic damage in the central nervous system by regulating synaptic stability. The present study was designed to investigate whether the SNK and SPAR signaling pathway was involved in Aβ-induced neurotoxicity in rat primary neurons. We measured mRNA and protein expression levels of SNK and SPAR in primary hippocampal neurons following Aβ treatment and used RNA interference to knockdown SNK to investigate the underlying mechanism. Expression of SNK and SPAR was altered by Aβ treatment, indicating that the SNK and SPAR signaling pathways may be involved in the damage to dendritic spines in hippocampal neurons induced by Aβ.
The signal-induced proliferation-associated (SIPA) protein family belongs to the RapGAP protein superfamily. Previous studies mainly focused on the expression and function of SIPA genes in vertebrate neuronal tissue. Only limited data about the embryonic expression pattern of the genes are currently available. Our study provides the first expression analysis of sipa1, sipa1l1, sipa1l2, and sipa1l3 during early development of the vertebrate organism Xenopus laevis. In silico, analysis revealed that all genes are highly conserved across species. Semi-quantitative RT-PCR experiments demonstrated that the RNA of all genes was maternally supplied. By whole mount in situ hybridization approaches, we showed that sipa1 is mainly expressed in various sensory organs, the respiratory and blood system, heart, neural tube, and eye. In contrast, sipa1l1 showed a broad expression during development in particular within the brain, somites, eye, and heart. Sipa1l2 was detected in the branchial arches, glomerulus, and the developing eye. In contrast, sipa1l3 revealed a tissue specific expression within the olfactory and otic vesicles, the cranial placodes and ganglia, neural tube, pronephros, retina, and lens. In summary, all sipa gene family members are expressed throughout the whole developing Xenopus organism and might play an important role during vertebrate early embryogenesis.
Lipids include sterols, mono- and diglycerides, phospholipids, among others. They operate as structural components of cell membranes (Vol. 1 – Chap. 7. Plasma Membrane; Tables 2.1 and 2.2), contributors of energy metabolism, participants of intracellular transport (Vol. 1 – Chap. 9. Intracellular Transport), and signaling molecules.
Protein tyrosine kinases (PTK), i.e., enzymes that catalyze the phosphorylation of Tyr residues of proteins. are mainly associated with growth factor signaling. They actually modulate multiple cellular events, such as differentiation, growth, metabolism, and apoptosis. On the other hand, protein serine/ threonine kinases are principally related to second messengers, such as cyclic nucleotides cAMP (Sect. 11.1) and cGMP (Sect. 11.2), lipidic and related mediators diacylglycerol and inositol trisphosphate (Chap. 2), and calmodulin (Sect. 11.5.3).
Mitogen-activated protein kinase (MAPK) modules are commonly used to transduce signals aimed at regulating cell fate (cell differentiation, proliferation, senescence, and death) and inflammation. They mediate responses to extracellular signals, such as hormones, growth factors, and cytokines, as well as stresses and intercellular interactions.
Protein phosphorylation is a common, reversible, post-translational modification of proteins (Vols.1 – Chap. 5. Protein Synthesis and 3 – Chap. 1. Signal Transduction). Except pseudophosphatases, phosphatases removes a phosphate group from their substrates. They thus antagonize kinases that attach phosphate groups to Ser, Thr, and Tyr residues in the same substrates using ATP. Phosphorylated residues SerP, ThrP, and TyrP account for about 86, 12, and 2% of the phosphoproteome, respectively. Mammalian genomes encode about 100 protein Tyr kinases and phosphatases, and about 100 and about 25 protein Ser/Thr kinases and phosphatases.
Guanosine triphosphatases intervene in: (1) signal transduction from the intracellular edge of the plasma membrane and intracellular domain of transmembrane receptors; (2) protein synthesis at the ribosome (Vol. 1 – Chap. 5. Protein Synthesis); (3) control of cell division (Vol. 2 – Chap. 2. Cell Growth and Proliferation); (4) proper protein folding; (5) translocation of proteins through the membrane of the endoplasmic reticulum; and (6) vesicular transport within the cell (Vol. 1 – Chap. 9. Intracellular Transport).
Other important signaling mediators include: (1) endogenous, gaseous, diffusible messengers, or gasotransmitters, such as carbon monoxide (CO; Sect. 10.2), nitric oxide (NO; Sect. 10.3), and hydrogen sulfide (H2S; Sect. 10.4), in addition to oxygen and carbon dioxide, as well as several reactive oxygen (ROS) and nitrogen species (RNS; Sect. 10.6), that act as intra- and intercellular regulators; (2) membrane-bound enzymatic complexes such as the reduced form of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase or NOx; Sect. 10.5); (3) some coregulators, such as (protein kinase) A-kinase-anchoring proteins (AKAP; Sect. 10.8.4) and annexins (Sect. 10.8.5); and (4) transcription factors involved in stress response, such as the proteic complex nuclear factor κ light-chain-enhancer of activated B cells (NFκB; Sect. 10.9.1), the heterodimer hypoxia-inducible factors (HIF; Sect. 10.9.2), and members of the Forkhead box (Fox; Sect. 10.9.3) and P53 families (Sect. 10.9.4; Table 10.1).
Numerous protein serine/threonine kinases operate in signal transduction, either as initiating nodes, such as receptor (RSTK; Vol. 3 – Chap. 8. Receptor Kinases), or as intermediate nodes of signaling cascades, non-receptor (intracellular; NRSTK) protein Ser/Thr kinases. Typically, second messengers activate protein Ser/Thr kinases, whereas extracellular signals stimulate protein Tyr kinases (Chap. 4). For example, IκB kinases (IKK) of IκB inhibitors of nuclear factor-κB are serine kinases (Sect. 10.9.1).
Multiple signaling processes are characterized by bursts of calcium ions that moves to specific locations for optimal activation of cell activity. Intracellular calcium regulates numerous protein functions in tiny cellular domains ( V1.2 channels form signaling clusters in plasmalemmal nanodomains. Fluorescence microscopy is aimed at imaging in real time communication within and between cells. Calcium fluxes through Ca\({}^{++}\) channels and gap junctions can be imaged once constitutive proteins (e.g., connexin) have been tagged with tetracysteines [1429].
Dual-specificity kinases (DSK; or Ser/Thr/Tyr kinases) phosphorylate their substrates on serine, threonine, and/or tyrosine residues. Dual-specificity kinases intervene in the regulation of cell growth, differentiation, and apoptosis. Dual-specificity kinases include, at least, members of the superfamily of mitogen-activated protein kinases as well as those of the family of glycogen synthase kinases.
Among kinases (or phosphotransferases) that transfer phosphate groups from donor molecules such as ATP or GTP to specific substrates include protein kinases that modify the activity of their proteic substrates. Kinases are used to transmit signals in cells. Other types of kinases act on amino acids, lipids, carbohydrates, and nucleotides, that are then used in signaling pathways or cell metabolism.
Small Rap guanosine-tri-phosphate (GTP)ases are crucially involved in many cellular processes, including cell proliferation, differentiation, survival, adhesion and movement. In line, it has been shown that Rap signalling is involved in various aspects of neuronal differentiation, like the establishment of neuronal polarity or axonal growth cone movement. Rap GTPases can be activated by a wide variety of external stimuli, and this is mediated by specific guanine nucleotide exchange factors (RapGEFs). Inactivation of RapGTP can be achieved with the aid of specific GTPase-activating proteins (RapGAPs). In the brain, the most prominent RapGAPs are Rap1GAP and those of the spine-associated RapGAP (SPAR) family. This latter family consists of three members (SPAR1-3), from which two of them, namely SPAR1 and 2, have been investigated in more detail. As such, the localization of RapGAPs is crucially important in regulating Rap signalling at various sites in the cell and, for both SPAR1 and 2, enrichment at synaptic sites has been demonstrated. In recent years particularly the role of SPAR1 in shaping dendritic spine morphology has attracted considerable interest. In this review we will summarize the described actions of different RapGAPs expressed in the brain, and we will focus in particular on the SPAR family members.
Spine-associated Rap guanosine triphosphatase-activating protein (SPAR) is an important regulator of activity-dependent remodeling of synapses. It is also critically involved in both mature dendritic spine formation and the maintenance of spine maturity. Glutamate is a major neurotransmitter of the brain, and is involved in all aspects of cognitive function, as it is the primary transmitter utilized by the cortical and hippocampal pyramidal neurons. Glutamate has also been associated with neuronal dendritic spine damage. The precise molecular mechanisms underlying dendritic spine damage following glutamate-induced neurotoxicity remain unknown. In the current study, we measured mRNA and protein expression levels of SPAR and serum-inducible kinase (SNK) in primary hippocampal neurons following glutamate treatment. Expression of SPAR and SNK was altered by glutamate treatment, indicating that the SPAR and SNK signaling pathways may be involved in the damage to dendritic spines in hippocampal neurons following excitotoxicity induced by glutamate.
Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.
Memory formation in the brain is thought to be depending upon long lasting plastic changes of synaptic contacts that require alterations on the transcriptional level. Here, we characterize LAPSER1, a putative cytokinetic tumor suppressor that binds directly to ProSAP2/Shank3 and the synaptic Rap-Gap protein SPAR1 as a novel postsynaptic density component. Postsynaptic LAPSER1 is in complex with all important members of the canonical Wnt pathway including beta-catenin. Upon N-methyl-D-aspartate receptor-dependent activation, LAPSER1 and beta-catenin comigrate from the postsynaptic density to the nucleus and induce the transcription and translation of known beta-catenin target genes, including Tcfe2a and c-Myc. The nuclear export and cytoplasmic redistribution of beta-catenin is tightly regulated by LAPSER1. We postulate a postsynaptic cross-talk between N-methyl-D-aspartate receptors and a LAPSER1-beta-catenin complex that results in a self-regulated, synaptic activity-dependent expression of beta-catenin target genes. This calls for a novel role of Tcfe2a and c-Myc in plastic changes of neural tissue.
The highly conserved RasGEF1 family of proteins contain a C-terminal CDC25-Ras exchange motif domain and an N-terminal RasGEF-N domain, and are of unknown function and specificity. Using purified RasGEF1A and RasGEF1B proteins, as well as Ras family proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. Although Rap2 was implicated in the regulation of cell adhesion, the establishment of cell morphology, and the modulation of synapses in neurons, no specific guanine nucleotide exchange factor for Rap2 was previously identified. Using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B.
Many studies in recent years suggest that schizophrenia is a synaptic disease that crucially involves a hypofunction of N-methyl-D-aspartate receptor-mediated signaling. However, at present it is unclear how these pathological processes are reflected in the protein content of the synapse. We have employed two-dimensional gel electrophoresis in conjunction with mass spectrometry to characterize and compare the synaptic proteomes of the human left dorsolateral prefrontal cortex in chronic schizophrenia and of the cerebral cortex of rats treated subchronically with ketamine. We found consistent changes in the synaptic proteomes of human schizophrenics and in rats with induced ketamine psychosis compared to controls. However, commonly regulated proteins between both groups were very limited and only prohibitin was found upregulated in both chronic schizophrenia and the rat ketamine model. Prohibitin, however, could be a new potential marker for the synaptic pathology of schizophrenia and might be causally involved in the disease process.
The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.
Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues,
as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2
gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against
the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation
and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation
revealed that most tuberin is found in a membrane/particulate (100,000 g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity
of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells.
Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin
leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.
We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation. In normal organs it is preferentially expressed in both fetal and adult lymphohematopoietic tissues. A Spa-1-encoded protein of 68 kDa is localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rap1 GTPase-activating protein (GAP), and a fusion protein of this domain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ras or Rho in vitro. Unlike the human Rap1 GAP, however, SpanN also exhibited GAP activity for Ran, so far the only known Ras-related GTPase in the nucleus. In the presence of serum, stable Spa-1 cDNA transfectants of NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NIH/Spa-1 cells were serum starved to be arrested in the G1/G0 phase of the cell cycle, however, they, unlike the control cells, exhibited progressive Spa-1 p68 accumulation, and following the addition of serum they showed cell death resembling mitotic catastrophes of the S phase during cell cycle progression. The results indicate that the novel nuclear protein Spa-1, with a potentially active Ran GAP domain, severely hampers the mitogen-induced cell cycle progression when abnormally and/or prematurely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with the Ran/RCC-1 system, which is involved in the coordinated nuclear functions, including cell division.
The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.
Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on
8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive
activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers
and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.
In order to stabilize changes in synaptic strength, neurons activate a program of gene expression that results in alterations of their molecular composition and structure. Here we demonstrate that Fnk and Snk, two members of the polo family of cell cycle associated kinases, are co-opted by the brain to serve in this program. Stimuli that produce synaptic plasticity, including those that evoke long-term potentiation (LTP), dramatically increase levels of both kinase mRNAs. Induced Fnk and Snk proteins are targeted to the dendrites of activated neurons, suggesting that they mediate phosphorylation of proteins in this compartment. Moreover, a conserved C-terminal domain in these kinases is shown to interact specifically with Cib, a Ca(2+)- and integrin-binding protein. Together, these studies suggest a novel signal transduction mechanism in the stabilization of long-term synaptic plasticity.
The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.
Numerous LOH and mutation analysis studies in different tumor tissues, including prostate, indicate that there are multiple tumor suppressor genes (TSGs) present within the human chromosome 8p21-22 and 10q23-24 regions. Recently, we showed that LZTS1 (or FEZ1), a putative TSG located on 8p22, has the potential to function as a cell growth modulator. We report here the cloning, gene organization, cDNA sequence characterization and expression analysis of LAPSER1, an LZTS1-related gene. This gene maps within a subregion of human chromosome 10q24.3 that has been reported to be deleted in various cancers, including prostate tumors, as frequently as the neighboring PTEN locus. The complete LAPSER1 cDNA sequence encodes a predicted protein containing various domains resembling those typically found in transcription factors (P-Box, Q-rich and multiple leucine zippers). LAPSER1 is expressed at the highest levels in normal prostate and testis, where multiple isoforms are seen, some of which are either undetectable or differentially expressed in some prostate tumor tissues and cell lines. Over-expression of LAPSER1 cDNA strongly inhibited cell growth and colony-forming efficiencies of most cancer cells assessed. Together these data suggest that LAPSER1 is another gene involved in the regulation of cell growth whose loss of function may contribute to the development of cancer.
Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.
Spa-1-like protein (SPAL) is a novel GTPase-activating protein (GAP) family protein. We generated anti-SPAL antibody, and examined localization of SPAL in the rat esophagus and heart by immunofluorescence microscopy. In the esophagus, SPAL was highly expressed in the spinous and granular layers and localized at the cell-cell border of the epithelial cells. SPAL in the spinous layer was almost colocalized with beta-catenin, an intracellular anchor protein of E-cadherin, while colocalization of SPAL with the tight junction protein ZO-1 was shown in the granular layer. In the heart, SPAL was localized at the intercalated disks, and there colocalized with both beta-catenin and ZO-1. In addition, much punctate localization of SPAL was seen along lateral borders of the cardiomyocytes, where beta-catenin and ZO-1 were absent. From these results, it is suggested that SPAL may regulate cell-cell adhesion in the rat esophagus and heart.
Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated
by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed
by Rap1GAP, a catalytically active fragment was expressed inEscherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 105-fold by Rap1GAP and has a k
catof 6 s−1 at 25 °C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active
site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducingV
max of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely
by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61.
The Rap1(G12V) mutant which in Ras reduces catalysis 106-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological
studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed
GTP-hydrolysis reaction on Rap1.
The PSD-95 family of proteins possesses multiple protein binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain. The PSD-95 proteins function as scaffolding proteins that link ion channels such as the N-methyl-d-aspartate-receptors (NMDA-Rs) with cytoskeletal networks and signalling molecules, thereby controlling synaptic plasticity and learning.
We found that the PSD-95 family proteins interact via their GK domains with SPA-1-like protein (SPAL), a GTPase-activating protein (GAP) that is specific for Rap1. SPAL was contained within the NMDA-R-PSD-95 complex, and co-localized with PSD-95 and NMDA-R at the synapses in cultured hippocampal neurones. Furthermore, NMDA stimulation induced the dephosphorylation of SPAL in cultured hippocampal neurones.
Our findings suggest that SPAL may be involved in the NMDA-mediated organization of cytoskeletal networks and signal transduction.
Trk receptors are a family of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT3 and NT4). Neurotrophin signaling through these receptors regulates cell survival, proliferation, the fate of neural precursors, axon and dendrite growth and patterning, and the expression and activity of functionally important proteins, such as ion channels and neurotransmitter receptors. In the adult nervous system, the Trk receptors regulate synaptic strength and plasticity. The cytoplasmic domains of Trk receptors contain several sites of tyrosine phosphorylation that recruit intermediates in intracellular signaling cascades. As a result, Trk receptor signaling activates several small G proteins, including Ras, Rap-1, and the Cdc-42-Rac-Rho family, as well as pathways regulated by MAP kinase, PI 3-kinase and phospholipase-C-gamma (PLC-gamma). Trk receptor activation has different consequences in different cells, and the specificity of downstream Trk receptor-mediated signaling is controlled through expression of intermediates in these signaling pathways and membrane trafficking that regulates localization of different signaling constituents. Perhaps the most fascinating aspect of Trk receptor-mediated signaling is its interplay with signaling promoted by the pan-neurotrophin receptor p75NTR. p75NTR activates a distinct set of signaling pathways within cells that are in some instances synergistic and in other instances antagonistic to those activated by Trk receptors. Several of these are proapoptotic but are suppressed by Trk receptor-initiated signaling. p75NTR also influences the conformations of Trk receptors; this modifies ligand-binding specificity and affinity with important developmental consequences.
Recent evidence has emphasized the importance of p38 mitogen-activated protein kinase (MAPK) in the induction of metabotropic
glutamate receptor (mGluR)-dependent long term depression (LTD) at hippocampal CA3-CA1 synapses. However, the cascade responsible
of mGluR to activate p38 MAPK and the signaling pathway immediately downstream from it to induce synaptic depression is poorly
understood. Here, we show that transient activation of group I mGluR with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) activates p38 MAPK through G protein βγ-subunit, small GTPase Rap1, and MAPK kinase 3/6
(MKK3/6), thus resulting in mGluR5-dependent LTD. Furthermore, our data clearly show that an accelerating AMPA receptor endocytosis
by stimulating the formation of guanyl nucleotide dissociation inhibitor-Rab5 complex is a potential downstream processing
of p38 MAPK activation to mediate DHPG-LTD. These results suggest an important role for Rap1-MKK3/6-p38 MAPK pathway in the
induction of mGluR-dependent LTD by directly coupling to receptor trafficking machineries to facilitate the loss of synaptic
AMPA receptors.
The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons, N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.
Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Recent studies have reported on the molecular mechanisms underlying dendritic spine (spine) dynamics. Because most of these studies investigated spine dynamics by overexpressing constitutively active or dominant-negative PSD (postsynaptic density) proteins in cultured mature neurons, the results represent the enlargement of mature spines or their return to an immature state. Here, we developed the technique of in utero electroporation to investigate spine dynamics. Using this technique, we demonstrated the suppression of spine maturation by the C-terminal variants of PSD-Zip70 in vitro and in vivo. Transient overexpression of the C terminus of PSD-Zip70 and knock-down of PSD-Zip70 also displayed the destabilization of mature spines. We further found the PSD-Zip70 and SPAR (spine-associated RapGAP) interaction via the short C-terminal region of PSD-Zip70 and the GK-binding domain of SPAR. In association with immature spines induced by overexpression of the PSD-Zip70 C terminus or knock-down of PSD-Zip70, SPAR lost its spine localization. Overexpression of the GK-binding domain of SPAR also induced to form immature spines without affecting the localization of PSD-Zip70 in the small heads of filopodial spines. Our results suggest that PSD-Zip70 in collaboration with SPAR is critically involved in spine maturity, especially in the mature spine formation and the maintenance of spine maturity.
Shank proteins assemble glutamate receptors with their intracellular signaling apparatus and cytoskeleton at the postsynaptic density. Whether Shank plays a role in spinogenesis and synaptogenesis remained unclear. Here, we report that knock-down of Shank3/prolinerich synapse-associated protein-2 by RNA interference reduces spine density in hippocampal neurons. Moreover, transgene expression of Shank 3 is sufficient to induce functional dendritic spines in aspiny cerebellar neurons. Transfected Shank protein recruits functional glutamate receptors, increases the number and size of synaptic contacts, and increases amplitude, frequency, and the AMPA component of miniature EPSCs, similar to what is observed during synapse developmental maturation. Mutation/deletion approaches indicate that these effects require interactions of Shank3 with the glutamate receptor complex. Consistent with this observation, chronic treatment with glutamate receptor antagonists alters maturation of the Shank3-induced spines. These results strongly suggest that Shank proteins and the associated glutamate receptors participate in a concerted manner to form spines and functional synapses.
ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density
(PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one
of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding
partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology
with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins.
ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3
co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with
a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization
of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the “Fezzins,”
which adds further complexity to the organization of the PSD protein network.
Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.
Remodeling of the endothelial cell (EC) cytoskeleton is central to many functions of the endothelium. The Rho family of small GTP-binding proteins have been identified as key regulators of F-actin cytoskeletal dynamics in a variety of cell types. They integrate signals from soluble mediators interacting with cytokine, growth factor tyrosine kinase, and G-protein–coupled receptors (GPCRs); as well as signals from cell–cell, and cell–matrix protein adhesion molecules. Recently, it has become appreciated that effector molecules downstream of Rho GTP-binding proteins also modulate several other well described cell signaling pathways. We review the role these molecules play in the cell, with a particular focus on the EC. HISTORY The Rho family of small GTP-binding proteins, which consists of 22 members, is part of the larger Ras GTP-binding protein superfamily. These approximately 21-kDa proteins cycle between inactive GDP-and active GTP-bound forms to act as a molecular switch in signal transduction pathways. The members of this family are grouped by virtue of a shared structural motif, the Rho insert loop, that is present in the GTPase domain and contributes to the binding specificity for downstream effector molecules (1,2). In addition to this shared structural feature, most Rho family members undergo post-translational modification to link farnesyl or geranylgeranyl groups to the cysteine in a CAAX motif at the C-terminus of the molecule. Subcellular localization of the molecule is directed by the lipid moiety and, in some family members, is also influenced by additional domains in the C-terminus.
The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.
The spatio-temporal pattern of expression of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) genes was analyzed in the postnatal developing cerebellar system of the rat by in situ hybridization histochemistry. Different ontogenetic patterns of expression were observed for these three genes. In agreement with previously published results (Neuron, 5 (1990) 501-509; Dev. Brain Res., 55 (1990) 288-292) we found that NT3 and LNGFR mRNA peaked early, during the first 2 postnatal weeks, whereas BDNF mRNA peaked later, around postnatal day 20, in the cerebellar cortex. High levels of NT3 mRNA were found in the internal granule cell layer as early as postnatal day 5. NT3 mRNA was also present in the external-premigratory granule cell layer at postnatal day 10. From postnatal day 5 on, LNGFR mRNA was present in the proliferative area of the external granule cell layer and in the Purkinje cells. NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation, suggesting a sequential stimulation of these two genes during this developmental process. LNGFR mRNA levels decreased along the same period. Although practically undetectable in the cerebellar granule cell layer in the first two postnatal weeks, BDNF mRNA was transiently expressed in the deep cerebellar nuclei during this time and it was very abundant in the inferior olivary system from postnatal day 5 on. LNGFR mRNA was transiently expressed in the inferior olivary system, in the first postnatal week. These data are discussed in relation to the coordinated postnatal maturation of the different cells of the cerebellar system.(ABSTRACT TRUNCATED AT 250 WORDS)
While target-derived neurotrophins are required for the survival of developing neurons in the PNS, the functions of neurotrophins in the CNS are unclear. Mice with a targeted gene deletion of brain-derived neurotrophic factor (BDNF) exhibit a wide-based gait. Consistent with this behavioral evidence of cerebellar dysfunction, there is increased death of granule cells, stunted growth of Purkinje cell dendrites, impaired formation of horizontal layers, and defects in the rostral-caudal foliation pattern. These abnormalities are accompanied by decreased Trk activation in granule and Purkinje cells of mutant animals, indicating that both cell types are direct targets for BDNF. These data suggest that BDNF acts as an anterograde or an autocrine-paracrine factor to regulate survival and morphologic differentiation of developing CNS neurons, and thereby affects neural patterning.
Stimulation of the intrinsic GTPase activity of GTP-binding proteins by GTPase-activating proteins (GAPs) is a basic principle of GTP-binding-protein downregulation. Recently, the molecular mechanism behind this reaction has been elucidated by studies on Ras and Rho, and their respective GAPs. The basic features involve stabilizing the existing catalytic machinery and supplementing it by an external arginine residue. This represents a novel mechanism for enzyme active-site formation.
Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.
The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1α, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, β-actin, α-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-d-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations.
Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.
The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.
Caldendrin is the founder member of a recently discovered family of calmodulin-like proteins, which are highly abundant in brain. In this study we examined the organization of the murine and human caldendrin gene as well as the expression pattern of transcripts for caldendrin and two novel splice variants. In addition the distribution of caldendrin in rat brain has been assessed by immunohistochemistry. Caldendrin is localized to the somatodendritic compartment of a subpopulation of mainly principal neurons in brain regions with a laminar organization and is present only at a subset of mature excitatory synapses. Caldendrin immunoreactivity (IR) is tightly associated with the cortical cytoskeleton, enriched in the postsynaptic density (PSD) fraction, and associates late during development with the synaptic cytomatrix. The expression is highly heterogenous within cortex, with highest levels of caldendrin IR in layer III of the piriform and layer II/III of the somatosensory cortex. The segregated cortical distribution to areas, which represent the most important primary sensory systems of the rodent brain, may reflect different requirements for dendritic Ca2+-signaling in these neurons. The presence of caldendrin in the PSD of distinct synapses may have important implications for Ca2+-modulated processes of synaptic plasticity.
The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.
Recent studies show that AMPA receptor (-R) trafficking is important in synaptic plasticity. However, the signaling controlling this trafficking is poorly understood. Small GTPases have diverse neuronal functions and their perturbation is responsible for several mental disorders. Here, we examine the small GTPases Ras and Rap in the postsynaptic signaling underlying synaptic plasticity. We show that Ras relays the NMDA-R and CaMKII signaling that drives synaptic delivery of AMPA-Rs during long-term potentiation. In contrast, Rap mediates NMDA-R-dependent removal of synaptic AMPA-Rs that occurs during long-term depression. Ras and Rap exert their effects on AMPA-Rs that contain different subunit composition. Thus, Ras and Rap, whose activity can be controlled by postsynaptic enzymes, serve as independent regulators for potentiating and depressing central synapses.
Typical members of the Ras superfamily of small monomeric GTP-binding proteins function as regulators of diverse processes by cycling between biologically active GTP- and inactive GDP-bound conformations. Proteins that control this cycling include guanine nucleotide exchange factors or GEFs, which activate Ras superfamily members by catalyzing GTP for GDP exchange, and GTPase activating proteins or GAPs, which accelerate the low intrinsic GTP hydrolysis rate of typical Ras superfamily members, thus causing their inactivation. Two among the latter class of proteins have been implicated in common genetic disorders associated with an increased cancer risk, neurofibromatosis-1, and tuberous sclerosis. To facilitate genetic analysis, I surveyed Drosophila and human sequence databases for genes predicting proteins related to GAPs for Ras superfamily members. Remarkably, close to 0.5% of genes in both species (173 human and 64 Drosophila genes) predict proteins related to GAPs for Arf, Rab, Ran, Rap, Ras, Rho, and Sar family GTPases. Information on these genes has been entered into a pair of relational databases, which can be used to identify evolutionary conserved proteins that are likely to serve basic biological functions, and which can be updated when definitive information on the coding potential of both genomes becomes available.
The Ras superfamily of small G proteins is remarkable for both its diversity and physiological functions. One member, Rap1, has been implicated in a particularly wide range of biological processes, from cell proliferation and differentiation to cell adhesion. But the diversity of Rap1 has lead to contradictory reports of its effects. Originally identified as an antagonist of Ras-induced transformation, Rap1 can oppose other actions of Ras including regulation of cell growth and differentiation, integrin-dependent responses and synaptic plasticity. Furthermore, recent evidence confirms that Rap1, like Ras, can activate the MAP kinase cascade (ERK) in several cell types. These diverse functions of Rap1 underscore that the activation and action of Rap1 are regulated by complex factors that are cell-type specific.
Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.
Synaptic plasticity involves the reorganization of synapses at the protein and the morphological levels. Here, we report activity-dependent
remodeling of synapses by serum-inducible kinase (SNK). SNK was induced in hippocampal neurons by synaptic activity and was
targeted to dendritic spines. SNK bound to and phosphorylated spine-associated Rap guanosine triphosphatase activating protein
(SPAR), a postsynaptic actin regulatory protein, leading to degradation of SPAR. Induction of SNK in hippocampal neurons eliminated
SPAR protein, depleted postsynaptic density–95 and Bassoon clusters, and caused loss of mature dendritic spines. These results
implicate SNK as a mediator of activity-dependent change in the molecular composition and morphology of synapses.
Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Galpha proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 A. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.
The small GTPases of the Ras superfamily mediate numerous biological processes through their ability to cycle between an inactive GDP-bound and an active GTP-bound form. Among the key regulators of GTPase cycling are the GTPase-activating proteins (GAPs), which stimulate the weak intrinsic GTP-hydrolysis activity of the GTPases, thereby inactivating them. Despite the abundance of GAPs and the fact that mutations in GAP-encoding genes underlie several human diseases, these proteins have received relatively little attention. Recent studies have addressed the regulatory mechanisms that influence GAP activity. So far, findings suggest that GAP activity is regulated by several mechanisms, including protein-protein interactions, phospholipid interactions, phosphorylation, subcellular translocation and proteolytic degradation.
The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.
The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.
Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.
Rap1 is a small GTP-binding protein that has been implicated in intracellular signaling and cytoskeletal control. Here, we show that Rap1 is expressed in rat cortical neurons and plays a critical role in dendritic development. Inhibition of Rap1 signaling either by expressing dominant negative mutant of Rap1 or Rap1GAP in cortical neurons reduced dendritic complexity. In contrast, expression of a constitutively active mutant of Rap1 (Rap1V12) induced dendritic growth and branching. Membrane depolarization, which induces dendritic growth via calcium influx, led to a rapid activation of Rap1 via cAMP and cGMP signaling. A CREB-dependent mechanism is involved in depolarization-induced dendritic growth in cortical neurons. Rap1 function contributed to depolarization induced CREB activation, and inhibition of CREB suppressed dendritic growth induced by Rap1V12. These observations identify Rap1 as a key mediator of calcium regulation of CREB-dependent transcription and dendritic development.
The small GTPase Rap1 is involved in several aspects of cell adhesion, including integrin-mediated cell adhesion and cadherin-mediated cell junction formation. Recently, several effector proteins for Rap1 have been identified providing a clear link between Rap1 and actin dynamics. Furthermore, evidence is accumulating that Rap1 functions in the spatial and temporal control of cell polarity.
The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.
The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.
Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.
With the aid of microarray and PCR analysis, this investigation sought expression profiles of BDNF-regulated genes in cultured mouse cerebellar granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Many of the BDNF-upregulated and downregulated genes identified were upregulated and downregulated, respectively, during cerebellar development. This developmental change was, at least partly, prevented in the TrkB receptor-deficient cerebellum. The BDNF-upregulated genes were distributed in either postmigratory or both premigratory and postmigratory granule cells at postnatal day 8 (P8) and were still present in mature granule cells at P21. In contrast, the BDNF-downregulated genes were predominantly expressed in premigratory granule cells at P8 and disappeared at P21. Furthermore, many of the BDNF-upregulated gene products are implicated in signaling cascades of N-methyl-D-aspartate receptors and MAP kinase. The results indicate that BDNF signaling plays a pivotal role in promoting gene expression in granule cell development and maturation.