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• W3133655105 startingPage "100517" @default.
• W3133655105 abstract "G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons. G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons. G protein–coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins and modulate a variety of physiological and pathological functions of the nervous system. Neurons are the most sophisticated cells with specialized morphology and compartmentalization in which most GPCRs are expressed in both presynaptic and postsynaptic membrane terminals where they are able to bind to respective neurotransmitters to activate cognate heterotrimeric G proteins or other signaling molecules which in turn activate downstream effectors, including ion channels, and thus control synaptic signaling and transmission (1Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (367) Google Scholar, 2Huang Y. Thathiah A. Regulation of neuronal communication by G protein-coupled receptors.FEBS Lett. 2015; 589: 1607-1619Crossref PubMed Scopus (66) Google Scholar, 3Weinberg Z.Y. Crilly S.E. Puthenveedu M.A. Spatial encoding of GPCR signaling in the nervous system.Curr. Opin. Cell Biol. 2019; 57: 83-89Crossref PubMed Scopus (14) Google Scholar). GPCRs are synthesized in the endoplasmic reticulum (ER). After being correctly folded and properly assembled, newly synthesized receptors can pass through the ER quality control system and export from the ER through the Golgi, along the axon and dendrites, to the synaptic compartments (4Valdes V. Valenzuela J.I. Salas D.A. Jaureguiberry-Bravo M. Otero C. Thiede C. Schmidt C.F. Couve A. Endoplasmic reticulum sorting and kinesin-1 command the targeting of axonal GABAB receptors.PLoS One. 2012; 7e44168Crossref PubMed Scopus (13) Google Scholar, 5Liebmann T. Kruusmagi M. Sourial-Bassillious N. Bondar A. Svenningsson P. Flajolet M. Greengard P. Scott L. Brismar H. Aperia A. A noncanonical postsynaptic transport route for a GPCR belonging to the serotonin receptor family.J. Neurosci. 2012; 32: 17998-18008Crossref PubMed Scopus (18) Google Scholar). Although previous studies have demonstrated that GPCR surface transport in neurons is regulated by specific motifs and interacting proteins (6Carrel D. Hamon M. Darmon M. Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting.J. Cell Sci. 2006; 119: 4276-4284Crossref PubMed Scopus (39) Google Scholar, 7Jolimay N. Franck L. Langlois X. Hamon M. Darmon M. Dominant role of the cytosolic C-terminal domain of the rat 5-HT1B receptor in axonal-apical targeting.J. Neurosci. 2000; 20: 9111-9118Crossref PubMed Google Scholar, 8Doly S. Shirvani H. Gata G. Meye F.J. Emerit M.B. Enslen H. Achour L. Pardo-Lopez L. Yang S.K. Armand V. Gardette R. Giros B. Gassmann M. Bettler B. Mameli M. et al.GABAB receptor cell-surface export is controlled by an endoplasmic reticulum gatekeeper.Mol. Psychiatry. 2016; 21: 480-490Crossref PubMed Scopus (34) Google Scholar, 9Al Awabdh S. Miserey-Lenkei S. Bouceba T. Masson J. Kano F. Marinach-Patrice C. Hamon M. Emerit M.B. Darmon M. A new vesicular scaffolding complex mediates the G-protein-coupled 5-HT1A receptor targeting to neuronal dendrites.J. Neurosci. 2012; 32: 14227-14241Crossref PubMed Scopus (24) Google Scholar, 10Carrel D. Masson J. Al Awabdh S. Capra C.B. Lenkei Z. Hamon M. Emerit M.B. Darmon M. Targeting of the 5-HT1A serotonin receptor to neuronal dendrites is mediated by Yif1B.J. Neurosci. 2008; 28: 8063-8073Crossref PubMed Scopus (52) Google Scholar, 11Zhang M. Huang W. Gao J. Terry A.V. Wu G. Regulation of alpha2B-adrenergic receptor cell surface transport by GGA1 and GGA2.Sci. Rep. 2016; 6: 37921Crossref PubMed Scopus (15) Google Scholar), the molecular mechanisms underlying the synaptic targeting and sorting of nascent GPCRs are poorly defined. Rab GTPases are the master regulators of vesicle-mediated membrane traffic in exocytic and endocytic pathways; they regulate a number of events involved in the development of central nervous system and neuronal functioning, such as neurite morphogenesis and outgrowth, axonal and dendritic transport, synaptic vesicle fusion and elimination, neurotransmitter release, and synaptic transmission (12Mori Y. Matsui T. Furutani Y. Yoshihara Y. Fukuda M. Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons.J. Biol. Chem. 2012; 287: 8963-8973Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Binotti B. Pavlos N.J. Riedel D. Wenzel D. Vorbruggen G. Schalk A.M. Kuhnel K. Boyken J. Erck C. Martens H. Chua J.J. Jahn R. The GTPase Rab26 links synaptic vesicles to the autophagy pathway.Elife. 2015; 4e05597Crossref PubMed Scopus (57) Google Scholar, 14Nakazawa H. Sada T. Toriyama M. Tago K. Sugiura T. Fukuda M. Inagaki N. Rab33a mediates anterograde vesicular transport for membrane exocytosis and axon outgrowth.J. Neurosci. 2012; 32: 12712-12725Crossref PubMed Scopus (43) Google Scholar, 15Mignogna M.L. D'Adamo P. Critical importance of RAB proteins for synaptic function.Small GTPases. 2018; 9: 145-157Crossref PubMed Scopus (28) Google Scholar, 16Kobayashi H. Fukuda M. Rab35 establishes the EHD1-association site by coordinating two distinct effectors during neurite outgrowth.J. Cell Sci. 2013; 126: 2424-2435Crossref PubMed Scopus (50) Google Scholar, 17Ng E.L. Tang B.L. Rab GTPases and their roles in brain neurons and glia.Brain Res. Rev. 2008; 58: 236-246Crossref PubMed Scopus (111) Google Scholar). Among more than 60 Rabs identified in mammals, some members are specific or enriched in neurons and perform neuron-specific functions, and ubiquitously expressed Rabs may have specialized functions in neurons (18Chan C.C. Scoggin S. Wang D. Cherry S. Dembo T. Greenberg B. Jin E.J. Kuey C. Lopez A. Mehta S.Q. Perkins T.J. Brankatschk M. Rothenfluh A. Buszczak M. Hiesinger P.R. Systematic discovery of Rab GTPases with synaptic functions in Drosophila.Curr. Biol. 2011; 21: 1704-1715Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Cherry S. Jin E.J. Ozel M.N. Lu Z. Agi E. Wang D. Jung W.H. Epstein D. Meinertzhagen I.A. Chan C.C. Hiesinger P.R. Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function.Elife. 2013; 2e01064Crossref PubMed Google Scholar). For instance, Rab3 and Rab27, two best-characterized neuron-specific Rabs, control synaptic vesicle exocytosis and the efficiency of neurotransmitter release (20Dulubova I. Lou X. Lu J. Huryeva I. Alam A. Schneggenburger R. Sudhof T.C. Rizo J. A Munc13/RIM/Rab3 tripartite complex: From priming to plasticity?.EMBO J. 2005; 24: 2839-2850Crossref PubMed Scopus (181) Google Scholar, 21Schluter O.M. Schmitz F. Jahn R. Rosenmund C. Sudhof T.C. A complete genetic analysis of neuronal Rab3 function.J. Neurosci. 2004; 24: 6629-6637Crossref PubMed Scopus (209) Google Scholar, 22Geppert M. Bolshakov V.Y. Siegelbaum S.A. Takei K. De Camilli P. Hammer R.E. Sudhof T.C. The role of Rab3A in neurotransmitter release.Nature. 1994; 369: 493-497Crossref PubMed Scopus (400) Google Scholar, 23Geppert M. Goda Y. Stevens C.F. Sudhof T.C. The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion.Nature. 1997; 387: 810-814Crossref PubMed Scopus (344) Google Scholar, 24Pavlos N.J. Gronborg M. Riedel D. Chua J.J. Boyken J. Kloepper T.H. Urlaub H. Rizzoli S.O. Jahn R. Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis.J. Neurosci. 2010; 30: 13441-13453Crossref PubMed Scopus (70) Google Scholar). Several Rabs have been shown to mediate agonist-induced GPCR endocytosis and the recycling of internalized receptors in neurons (25Wang G. Wu G. Small GTPase regulation of GPCR anterograde trafficking.Trends Pharmacol. Sci. 2012; 33: 28-34Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Esseltine J.L. Ribeiro F.M. Ferguson S.S. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.J. Neurosci. 2012; 32: 16933A-16942ACrossref PubMed Scopus (31) Google Scholar, 27Holmes K.D. Babwah A.V. Dale L.B. Poulter M.O. Ferguson S.S. Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases.J. Neurochem. 2006; 96: 934-949Crossref PubMed Scopus (72) Google Scholar). However, virtually nothing is known about the function and regulation of the Rab family in the transport of newly synthesized GPCRs to dendrites and synapses. To address these issues, in this study, we determine the role of Rab43 in the dendritic and synaptic transport of the prototypic family A α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR). The α2-AR and mAChR have three (α2A-, α2B-, and α2C-AR) and five subtypes (M1R–M5R), respectively, and all play important roles in the central and peripheral nervous systems. As compared with many other secretory Rab GTPases, the function of Rab43 is relatively poorly defined. Previous studies have shown that Rab43 is important for Golgi structure (28Haas A.K. Yoshimura S. Stephens D.J. Preisinger C. Fuchs E. Barr F.A. Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells.J. Cell Sci. 2007; 120: 2997-3010Crossref PubMed Scopus (162) Google Scholar, 29Cox J.V. Kansal R. Whitt M.A. Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi.Mol. Biol. Cell. 2016; 27: 1834-1844Crossref PubMed Scopus (12) Google Scholar), ER–Golgi transport (30Dejgaard S.Y. Murshid A. Erman A. Kizilay O. Verbich D. Lodge R. Dejgaard K. Ly-Hartig T.B. Pepperkok R. Simpson J.C. Presley J.F. Rab18 and Rab43 have key roles in ER-Golgi trafficking.J. Cell Sci. 2008; 121: 2768-2781Crossref PubMed Scopus (120) Google Scholar, 31Li C. Wei Z. Fan Y. Huang W. Su Y. Li H. Dong Z. Fukuda M. Khater M. Wu G. The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.Cell Rep. 2017; 21: 1089-1101Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), retrograde surface–Golgi transport (28Haas A.K. Yoshimura S. Stephens D.J. Preisinger C. Fuchs E. Barr F.A. Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells.J. Cell Sci. 2007; 120: 2997-3010Crossref PubMed Scopus (162) Google Scholar), phagosome maturation (32Seto S. Tsujimura K. Koide Y. Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes.Traffic. 2011; 12: 407-420Crossref PubMed Scopus (141) Google Scholar), and antigen cross-presentation by dendritic cells (33Kretzer N.M. Theisen D.J. Tussiwand R. Briseno C.G. Grajales-Reyes G.E. Wu X. Durai V. Albring J. Bagadia P. Murphy T.L. Murphy K.M. RAB43 facilitates cross-presentation of cell-associated antigens by CD8alpha+ dendritic cells.J. Exp. Med. 2016; 213: 2871-2883Crossref PubMed Scopus (49) Google Scholar). Here, we demonstrate that Rab43 mediates the dendritic and postsynaptic delivery of some but not all GPCRs, and the function of Rab43 in the sorting of different GPCRs is dictated by direct interaction and in a neuronal cell–specific manner. Our data also provide direct evidence implicating distinct biosynthetic pathways that deliver different GPCRs to the functional destinations in neurons. As an initial approach to study the possible function of Rab43 in regulating the anterograde transport of newly synthesized GPCRs in neurons, we measured the effect of lentiviral expression of dominant-negative guanine nucleotide-deficient mutant Rab43N131I on the surface expression of endogenous α2-AR and mAChR in primary cortical neurons in radioligand binding assays. Expression of Rab43N131I markedly reduced the surface number of α2-AR by approximately 55% as compared with neurons infected with control viruses (Fig. 1, A and B). Surprisingly, Rab43N131I expression had no significant effect on the surface expression of mAChR (Fig. 1B). In contrast, treatment with brefeldin A (BFA), which disrupts the Golgi structure and thus blocks the ER-to-Golgi transport, similarly inhibited the surface expression of both α2-AR and mAChR by 40 to 60% (Fig. 1C). To define if Rab43 could differentially affect the concomitant function of α2-AR and mAChR, we used the activation of mitogen-activated protein kinases, specifically extracellular signal–regulated kinase 1 and 2 (ERK1/2), as a readout. Although α2-AR and mAChR each have multiple subtypes which couple to distinct heterotrimeric G proteins, they are all able to activate ERK1/2. Consistent with its effects on the surface receptor expression measured in radioligand binding assays, lentiviral expression of Rab43N131I strongly inhibited ERK1/2 activation in response to stimulation with the α2-AR agonist UK14304, without altering ERK1/2 activation by the mAChR agonist oxotremorine-m (Oxo-M) (Fig. 1, D and E). These data demonstrate that the surface expression of α2-AR and mAChR, as well as their signaling, is differentially regulated by Rab43 in neurons. We next determined the role of Rab43 in the dendritic and postsynaptic transport of α2-AR and mAChR, specifically the α2B-AR and M3 mAChR (M3R) subtypes, in primary cultures of hippocampal neurons by confocal imaging. We first determined if expression of Rab43N131I could alter the general morphology of neurons. As accessed by the DsRed and GFP signals, expression of DsRed-tagged Rab43N131I had no obvious effect on the general morphology of hippocampal neurons compared with neurons transfected with DsRed vectors (Fig. 2, A and B). The spine length, width, and density were very much the same in neurons expressing DsRed and Rab43N131I (Fig. 2, B and C). Consistent with radioligand-binding data, Rab43N131I significantly reduced the dendritic expression of α2B-AR by approximately 40%, but had no effect on M3R expression at dendrites (Fig. 2, D–F). Rab43N131I also significantly attenuated the expression of α2B-AR at the dendritic spines by about 35%, whereas M3R expression at the dendritic spines was not affected (Fig. 2, G–I). We then studied the effect of Rab43KO by the CRISPR–Cas9 system on the dendritic and postsynaptic trafficking of α2B-AR and M3R in the hippocampal neurons. Owing to the lack to Rab43 antibodies detecting endogenous Rab43 in primary neurons, the efficiency of Rab43KO was tested by using transient expression of GFP-tagged rat Rab43 in human embryonic kidney 293 (HEK293) cells. The expression of rat Rab43 was reduced by more than 90% in cells transfected with Rab43KO plasmids targeting mouse Rab43 as compared with cells transfected with control vectors (Fig. 3A), suggesting that CRISPR–Cas9 KO plasmids targeting mouse Rab43 can deplete rat Rab43. In hippocampal neurons, similar to the effects of Rab43N131I, CRISPR–Cas9–mediated Rab43KO markedly inhibited the dendritic presentation of α2B-AR, but not M3R (Fig. 3, B–D). Rab43KO by CRISPR–Cas9 also reduced the expression of α2B-AR, but not M3R, at the dendritic spines (Fig. 3, E–G). To further confirm the role of Rab43 in the postsynaptic transport, hippocampal neurons were transfected with red fluorescent protein–tagged PSD95, GFP-carrying Rab43KO plasmids, and yellow fluorescent protein–tagged α2B-AR or M3R, and receptor expression at postsynapses was measured by using PSD95 as a marker. Postsynaptic expression of α2B-AR was markedly reduced, whereas M3R expression remained unaffected in Rab43KO neurons as compared with neurons transfected with control vectors (Fig. 3, H–J). These data demonstrate distinct functions of Rab43 in the forward transport of α2B-AR and M3R to the neuronal dendrites and postsynapses. We next compared the effects of Rab43 on the surface transport of α2-AR and mAChR in human-derived neuroblastoma SHSY5Y cells and rat renal tubular epithelial NRK49F cells. Similar to the results observed from primary cultures of neurons, Rab43N131I expression inhibited the total surface expression of endogenous α2-AR in both SHSY5Y and NRK49F cells by greater than 50%, whereas Rab43N131I only attenuated the surface number of endogenous mAChR in NRK49F cells, but not in SHSY5Y cells, as measured by radioligand binding (Fig. 4A). In contrast, BFA treatment significantly suppressed the surface expression of α2-AR and mAChR in both cell types (Fig. 4B). In bioluminescence resonance energy transfer (BRET) assays to measure receptor surface expression, Rluc-tagged α2B-AR or M3R was expressed together with venus-tagged kRas and Rab43 mutants. In addition to Rab43N131I, inactive GDP-bound Rab43T32N mutant was also used. The net BRET signal between α2B-AR and kRas was markedly inhibited by both Rab43 mutants, whereas the BRET signal between M3R and kRas was not affected by the Rab43 mutants in SHSY5Y cells (Fig. 4C). Confocal microscopy showed that, as expected, both α2B-AR and M3R were robustly expressed at the cell surface in SHSY5Y and NRK49F cells transfected with control vectors. In Rab43N131I-expressing cells, α2B-AR was unable to transport to the cell surface and arrested in the perinuclear regions in both cell types, whereas M3R retained its ability to sufficiently move to the cell surface in SHSY5Y cells, but not in NRK49F cells (Fig. 4D). We then determined the effect of CRISPR–Cas9–mediated Rab43KO on the subcellular localization of α2B-AR and M3R. Rab43KO plasmids targeting human Rab43 were tested using GFP-tagged human Rab43 in HEK293 cells (Fig. 5A). Similar to Rab43N131I, Rab43KO arrested α2B-AR in the perinuclear regions in both SHSY5Y and NRK49F cells, whereas M3R was arrested only in NRK49F cells, but not in SHSY5Y cells (Fig. 5B). We then sought to determine if single-guide RNA (sgRNA)-resistant Rab43 plasmids could rescue the effect of CRISPR–Cas9–mediated Rab43KO on the surface expression of M3R. For this purpose, human Rab43 plasmids were transfected together with mouse Rab43KO plasmids in NRK49F cells. Human Rab43 expression was not affected by mouse Rab43KO plasmids (Fig. 5C). Expression of human Rab43 effectively rescued the surface expression of cyan fluorescent protein (CFP)-tagged M3R in NRK49F cells expressing mouse Rab43KO plasmids (Fig. 5D). These data demonstrate that different actions of Rab43 on the surface transport of mAChR and specific M3R subtype are neuronal cell type specific. As our previous studies have shown that α2B-AR directly interacts with Rab43 via the third intracellular loop (ICL3), to elucidate the molecular mechanisms underlying the selective regulation of α2B-AR and M3R trafficking by Rab43 in neurons and neuronal ells, we compared Rab43 interaction with the ICL3 of both receptors in glutathione-S-transferase (GST) fusion protein pull-down assays. α2B-AR and M3R possess relatively large ICL3, containing 165 and 229 residues, respectively, which were generated as GST fusion proteins. Consistent with our previous data, the α2B-AR ICL3 strongly bound Rab43. In contrast, the M3R ICL3 binding to Rab43 was negligible (Fig. 6A). We then generated the chimeric receptor M3-2B in which the M3R ICL3 was substituted with the α2B-AR ICL3 (Fig. 6B) and measured the possible interactions between Rab43 and individual receptors in live cells in BRET assays. The net BRET signals observed between Rab43 and α2B-AR and between Rab43 and M3-2B were similar, but the net BRET signal between Rab43 and M3R was almost undetectable (Fig. 6C). These data suggest that the α2B-AR ICL3 can confer its interaction with Rab43 to M3R. We next determined the effect of Rab43 on M3-2B transport in cell lines and primary neurons. In contrast to M3R which is irresponsive to Rab43, the surface expression of M3-2B was inhibited by Rab43N131I and Rab43KO in SHSY5Y cells as measured by intact cell radioligand binding (Fig. 6D). Confocal microscopy showed that M3-2B was strongly expressed at the surface which was clearly impeded by Rab43N131I or Rab43KO in both SHSY5Y and NRK49F cells (Fig. 6, E and F). The intracellularly accumulated M3-2B was extensively colocalized with calregulin, an ER marker (Fig. 6, G and H). These data suggest that Rab43 is able to control M3-2B transport to the cell surface, specifically its export from the ER. In primary hippocampal neurons, M3-2B was expressed at dendrites and spines. Similar to the results observed in cells, M3-2B expression at dendrites (Fig. 7, A–C) and spines (Fig. 7, D–F) was significantly inhibited by Rab43N131I and Rab43KO. Furthermore, the inhibitory effects of Rab43N131I and Rab43KO on the transport of M3-2B were similar (Fig. 7, C and F). These data demonstrate that the α2B-AR ICL3 controls not only M3-2B interaction with Rab43 but also its Rab43-dependent trafficking. As the first study on the functions of the Rab family GTPases in the anterograde dendritic and synaptic delivery of GPCRs, we have focused on Rab43 which was recently demonstrated to regulate GPCR trafficking in the early secretory pathway in cell (31Li C. Wei Z. Fan Y. Huang W. Su Y. Li H. Dong Z. Fukuda M. Khater M. Wu G. The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.Cell Rep. 2017; 21: 1089-1101Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The most important finding presented in this article is that, through inhibiting the function of endogenous Rab43 by expression of dominant-negative mutants and CRISPR–Cas9–mediated KO, we have identified Rab43 as an important regulator in the anterograde trafficking of some but not all GPCRs in neurons. By using radioligand binding of intact live neurons to quantify the exact surface receptor number, we have demonstrated that Rab43 selectively regulates the total surface expression of endogenous α2-AR, but not mAChR, in primary neurons. By confocal microscopy to directly visualize the localization of fluorescence-tagged individual receptor subtypes, we have shown that the dendritic and postsynaptic expression of α2B-AR, but not M3R, depends on the normal function of Rab43. Such selective actions of Rab43 toward different GPCRs are also reflected by receptor-mediated signaling measured by ERK1/2 activation. These data provide direct evidence indicating novel functions for Rab43 in mediating GPCR transport to dendrites and synapses, as well as their sorting from one another in neurons. However, we cannot exclude the possibility that Rab43 may regulate the trafficking of other signaling molecules involved in receptor-mediated signal transduction pathways, which may also contribute to the observed abnormal signaling, and the role of Rab43 in the trafficking of other α2-AR and mAChR subtypes remains unknown. Similar to the results observed in primary neurons, Rab43 affects the surface expression of endogenous α2-AR and specific α2B-AR subtype, but not endogenous mAChR and M3R subtype, in neuronal SHSY5Y cells. In contrast, the surface transport of both receptors requires Rab43 in non-neuronal NRK49F cells. These data suggest that the sorting function of Rab43 in the trafficking of distinct GPCRs along the biosynthetic pathways is neuronal cell specific. These data also imply that trafficking itineraries of a GPCR (e.g., M3R) may differ in neurons/neuronal cells and non-neuronal cells. Although GPCRs share a common structural topology, how they are sorted into distinct transport pathways is poorly understood. Our previous studies suggest that Rab43 interaction may separate GPCRs from non-GPCR plasma membrane proteins (31Li C. Wei Z. Fan Y. Huang W. Su Y. Li H. Dong Z. Fukuda M. Khater M. Wu G. The GTPase Rab43 controls the anterograde ER-Golgi trafficking and sorting of GPCRs.Cell Rep. 2017; 21: 1089-1101Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Our results presented here have demonstrated that the M3R chimera containing the Rab43-binding domain identified in α2B-AR interacts with Rab43 and its dendritic and postsynaptic transport in primary neurons and the surface transport in neuronal cells depend on Rab43. These data indicate that the Rab43-binding domain is able to effectively convert GPCR transport from a Rab43-independent pathway into a Rab43-dependent pathway and that the sorting function of Rab43 is mediated through its direct interaction with the receptors. An important implication of this study is that different GPCRs may use distinct pathways (e.g., Rab43-dependent and Rab43-independent pathways) to transport from the cell body to dendrites and from the dendritic shaft to spines. It is possible that M3R transport to dendrites is mediated through lateral diffusion as suggested for most GPCRs (5Liebmann T. Kruusmagi M. Sourial-Bassillious N. Bondar A. Svenningsson P. Flajolet M. Greengard P. Scott L. Brismar H. Aperia A. A noncanonical postsynaptic transport route for a GPCR belonging to the serotonin receptor family.J. Neurosci. 2012; 32: 17998-18008Crossref PubMed Scopus (18) Google Scholar), which is independent of Rab43, whereas α2B-AR uses secretory vesicles to move forward to dendrites as suggested for serotonin 1B receptor (5HT1B) (5Liebmann T. Kruusmagi M. Sourial-Bassillious N. Bondar A. Svenningsson P. Flajolet M. Greengard P. Scott L. Brismar H. Aperia A. A noncanonical postsynaptic transport route for a GPCR belonging to the serotonin receptor family.J. Neurosci. 2012; 32: 17998-18008Crossref PubMed Scopus (18) Google Scholar), which is dependent on Rab43. It is interesting to note that, in addition to exist in the soma, the ER is distributed throughout the cytoplasm in dendrites, the Golgi forms discrete structures called “Golgi outposts” in the longest dendrite, and coat protein complex II (COPII) vesicles that exclusively mediate the ER export of newly synthesized molecules, including GPCRs (34Dong C. Zhou F. Fugetta E.K. Filipeanu C.M. Wu G. Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase.Cell Signal. 2008; 20: 1035-1043Crossref PubMed Scopus (32) Google Scholar), are formed in distal dendritic branches (35Aridor M. Guzik A.K. Bielli A. Fish K.N. Endoplasmic reticulum export site formation and function in dendrites.J. Neurosci. 2004; 24: 3770-3776Crossref PubMed Scopus (76) Google Scholar). The dendritic ER, Golgi outposts, and COPII vesicles all participate in the local delivery of plasma membrane receptors (35Aridor M. Guzik A.K. Bielli A. Fish K.N. Endoplasmic reticulum export site formation and function in dendrites.J. Neurosci. 2004; 24: 3770-3776Crossref PubMed Scopus (76) Google Scholar, 36Hanus C. Ehlers M.D. Secretory outposts for the local processing of membrane cargo in neuronal dendrites.Traffic. 2008; 9: 1437-1445Crossref PubMed Scopus (89) Google Scholar, 37Ramirez O.A. Couve A. The endoplasmic reticulum and protein trafficking in dendrites and axons.Trends Cell Biol. 2011; 21: 219-227Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 38Cui-Wang T. Hanus C. Cui T. Helton T. Bourne J. Watson D. Harris K.M. Ehlers M.D. Local zones of endoplasmic reticulum complexity confine cargo in neuronal dendrites.Cell. 2012; 148: 30" @default.
• W3133655105 created "2021-03-15" @default.
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• W3133655105 date "2021-01-01" @default.
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• W3133655105 title "Rab43 GTPase directs postsynaptic trafficking and neuron-specific sorting of G protein–coupled receptors" @default.
• W3133655105 cites W1483996398 @default.
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