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| Melanocortin receptor accessory protein 2 (MRAP2) variants form proper homodimers and interact with melanocortin-4 receptor (MC4R). (A-F) Coimmunoprecipitation (IP) of the 2FLAG-tagged and 3HA-tagged MRAP2 variants I, II, III, IV, V, and VI. The order of transfection in each group is from left to right: 2FLAG-tagged variant transfected only, 3HA-tagged variant transfected only, and 2FLAG-tagged and 3HA-tagged variants co-transfected. (G) Immunoprecipitation and Western blot detection of 3HA-D2dr, 3HA-MC4R, and 2Flag-MRAP2 variants or RAMP3. MRAP2 variants and RAMP3 were detected using mouse anti-Flag antibody. IB, immunoblotting, beads only, no antibody was used for the IP.
Source publication
The melanocortin receptors are defined as a series of vital pharmaceutical targets to regulate neuronal appetite and maintain controllable body weight for mammals and teleosts. Melanocortin receptor accessory protein 2 (MRAP2) functions as an essential accessory player that modulates the surface translocation and binding to a variety of endogenous...
Contexts in source publication
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... homodimerization of these variants, suggesting a robust interaction between these variant monomers (Figures 2A-F). Subsequently, we co-transfected the mMC4R and mMRAP2 plasmids into HEK293 cells to examine the interaction of the MRAP2 variants with their G-protein-coupled receptor (GPCR) targets. ...
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... we co-transfected the mMC4R and mMRAP2 plasmids into HEK293 cells to examine the interaction of the MRAP2 variants with their G-protein-coupled receptor (GPCR) targets. Co-immunoprecipitation (co-IP) assay exhibited the proper interaction of all six MRAP2 variants with MC4R in vitro, suggesting that the alteration of the interior orientation of each domain did not affect the interaction between these mutant mMRAP2 and mMC4R ( Figure 2G). RAMP3 (another singletransmembrane accessory protein) was utilized as a control group and showed no interplay with MC4R ( Figure 2G). ...
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... (co-IP) assay exhibited the proper interaction of all six MRAP2 variants with MC4R in vitro, suggesting that the alteration of the interior orientation of each domain did not affect the interaction between these mutant mMRAP2 and mMC4R ( Figure 2G). RAMP3 (another singletransmembrane accessory protein) was utilized as a control group and showed no interplay with MC4R ( Figure 2G). Meanwhile, co-IP of the dopamine receptor D2 with MRAP2 excluded nonspecific binding and immunoprecipitation (IP) artifacts, and b-actin was recruited as an internal reference for all co-IP analyses. ...
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... maximal cAMP response was enhanced when co-transfected with variants II, IV, and VI (Figures 3C, E, G), while variants I, III, and V inhibited the EC 100 stimulation level of a-MSH compared to that of wtMRAP2 ( Figures 3B, D, F). Furthermore, the EC 50 value was decreased in the presence of variants I-III, V, and VI, indicating the increased sensitivity of MC4R to a-MSH (Tables 1 and 2). The constitutive activity and maximum response of variants I (transmembrane region inversion) and III (C-terminal inversion) were both reduced. ...
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... studies have reported that MC4R locates at the cell membrane and colocalize with MRAP2 in live cells (23,24,26). Here, the steadily detectable fluorescence in Figure 7 indicated the co-localization of MC4R and MRAP2 or its variants and varified the direct interaction and formation of tight functional protein complexes, as detected in Figure 2. There was no significant difference in the fluorescence density between the dimeric groups, as seen in Figure 6B. ...
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... the fluorescence of the yellow fluorescent protein (YFP) between variant VI and MC4R showed a significant reduction compared with that of the other groups ( Figure 7B). These data were also consistent with the co-IP results in Figure 2G. ...
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... reversed the amino acid sequence of each interior domain to investigate the function of each MRAP2 mutant (Figure 1). Like wtMRAP2, all variants could interact with itself, suggesting that the internal transposition did not affect the homodimerization of MRAP2 in vitro (Figures 2A-F). The replacement of the C-terminal with the inverted N-terminal could still form a dimeric topology, indicating that the essential motif required for dimer formation should be within the N-terminal region of mMRAP2. ...
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... which indicated that the mouse MRAP2 could form both antiparallel and parallel dimers as well. Additionally, the presence of a "doublet band" for MRAP2 variant V indicated that the N-terminal asparagine residue existed in both glycosylated and unglycosylated forms ( Figure 2E). Co-IP was utilized to detect the interaction between MC4R and the MRAP2 variants ( Figure 2G). ...
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... the presence of a "doublet band" for MRAP2 variant V indicated that the N-terminal asparagine residue existed in both glycosylated and unglycosylated forms ( Figure 2E). Co-IP was utilized to detect the interaction between MC4R and the MRAP2 variants ( Figure 2G). As expected, we found that all MRAP2 variants interacted with MC4R, but with different intensities. ...
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... expected, we found that all MRAP2 variants interacted with MC4R, but with different intensities. Moreover, dopamine receptor D2, as a negative GPCR control, showed no interaction with all MRAP2 variants ( Figure 2G). Like many other vertebrates, we confirmed that mMC4R was capable of interacting with wtmMRAP2 and all six variants in vitro, as shown by BiFC (Figure 7). ...
Citations
... As shown in Figures 7 and S9, MRAP proteins showed a distinct effect on the cell surface expression of GPCRs. Consistent with the results in previous studies, 34,40 we found that both MRAP1 and MRAP2 significantly reduced the membrane transport of MC4R. We found that MRAP1 increased the membrane trafficking of most GPCRs, while MRAP2 decreased their surface expression. ...
Background:
The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown.
Methods:
Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs.
Results:
36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner.
Conclusions:
Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.
... First, 2xFlag tagged reversed mrap2a/b and 3xHA-mc4r were transfected into HEK293T cells and co-immunoprecipitation (CoIP) assays found that RMrap2a/b interacted with zMc4r ( Figure 1D,E) and that RMRAP2 also formed a protein complex with mMC4R (−37 kDa) ( Figure 1F). Receptor activity modifying protein 3 (RAMP3,~17 kDa) was utilized as a negative control ( Figure 1C,G) because RAMP3 functioned as a single transmembrane protein like MRAP2, but it could not interact with MC4R [37]. Some dispersive bands ranging from 60 to 80 kDa were seen on the Western blot, indicating the existence of MC4R polymers ( Figure 1C-F). ...
As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling.
The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors, but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R-MC5R) and the glucagon receptor (GCGR), respectively. Especially, MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at the atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell (Krishna Kumar et al., 2023) unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research (Luo et al., 2023) of the adrenocorticotropic hormone (ACTH)-bound MC2R–Gs–MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, and the recent structural investigation of MRAP-MC2R and RAMP-GCGR functional complex and the expanded identification of new GPCR partners of MRAP proteins. In-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR associated human disorders.
Somatostatin receptors (SSTRs) are G protein-coupled receptors (GPCRs) known to regulate exocrine secretion, neurotransmission, and inhibit endogenous cell proliferation. SSTR subtypes (SSTR1-SSTR5) exhibit homo- or heterodimerization with unique signaling characteristics. Melanocortin receptor accessory protein 1 (MRAP1) functions as an allosteric modulator of melanocortin receptors and some other GPCRs. In this study, we investigated the differential interaction of MRAP1 and SSTRs and examined the pharmacological modulation of MRAP1 on mouse SSTR2/SSTR3 and SSTR2/SSTR5 heterodimerization in vitro. Our results show that the mouse SSTR2 forms heterodimers with SSTR3 and SSTR5 and that MRAP1 selectively interacts with SSTR3 and SSTR5 but not SSTR2. The interactive binding sites of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 locate on SSTR3 and SSTR5 but not SSTR2. The binding sites of MRAP1 to SSTR3 are extensive, while the ones of SSTR5 are restricted on transmembrane region six and seven. The heterodimerization of mouse SSTR2, SSTR3, and SSTR5 can be modulated by binding protein in addition to an agonist. Upregulation of extracellular signal-regulated kinases phosphorylation, p27Kip1, and increased cell growth inhibition with the co-expression of SSTR2/SSTR3 or SSTR2/SSTR5 with MRAP1 suggest a regulatory effect of MRAP1 on anti-proliferative response of two SSTR heterodimers. Taken together, these results provide a new insight of MRAP1 on the maintenance and regulation of mouse SSTR dimers which might be helpful to better understand the molecular mechanism involving SSTRs in tumor biology or other human disorders.
