Specific motifs mediate post-synaptic and surface transport of G protein-coupled receptors.
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ABSTRACT:
SUBMITTER: Xu X
PROVIDER: S-EPMC8728401 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that alpha(2B)-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the alpha(2B)-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.
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Project description:The role of Gαs in G protein-coupled receptor (GPCR) signalling at the cell surface is well established. Recent evidence has revealed the presence of Gαs on endosomes and its capacity to elicit GPCR-promoted signalling from this intracellular compartment. Here, we report an unconventional role for Gαs in the endocytic sorting of GPCRs to lysosomes. Cellular depletion of Gαs specifically delays the lysosomal degradation of GPCRs by disrupting the transfer of GPCRs into the intraluminal vesicles (ILVs) of multivesicular bodies. We show that Gαs interacts with GPCR-associated binding protein-1 (GASP1) and dysbindin, two key proteins that serve as linkers between GPCRs and the endosomal-sorting complex required for transport (ESCRT) machinery involved in receptor sorting into ILVs. Our findings reveal that Gαs plays a role in both GPCR signalling and trafficking pathways, providing another piece in the intertwining molecular network between these processes.
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Project description:Neuropeptides play pivotal roles in various biological events in the nervous, neuroendocrine, and endocrine systems, and are correlated with both physiological functions and unique behavioral traits of animals. Elucidation of functional interaction between neuropeptides and receptors is a crucial step for the verification of their biological roles and evolutionary processes. However, most receptors for novel peptides remain to be identified. Here, we show the identification of multiple G protein-coupled receptors (GPCRs) for species-specific neuropeptides of the vertebrate sister group, Ciona intestinalis Type A, by combining machine learning and experimental validation. We developed an original peptide descriptor-incorporated support vector machine and used it to predict 22 neuropeptide-GPCR pairs. Of note, signaling assays of the predicted pairs identified 1 homologous and 11 Ciona-specific neuropeptide-GPCR pairs for a 41% hit rate: the respective GPCRs for Ci-GALP, Ci-NTLP-2, Ci-LF-1, Ci-LF-2, Ci-LF-5, Ci-LF-6, Ci-LF-7, Ci-LF-8, Ci-YFV-1, and Ci-YFV-3. Interestingly, molecular phylogenetic tree analysis revealed that these receptors, excluding the Ci-GALP receptor, were evolutionarily unrelated to any other known peptide GPCRs, confirming that these GPCRs constitute unprecedented neuropeptide receptor clusters. Altogether, these results verified the neuropeptide-GPCR pairs in the protochordate and evolutionary lineages of neuropeptide GPCRs, and pave the way for investigating the endogenous roles of novel neuropeptides in the closest relatives of vertebrates and the evolutionary processes of neuropeptidergic systems throughout chordates. In addition, the present study also indicates the versatility of the machine-learning-assisted strategy for the identification of novel peptide-receptor pairs in various organisms.
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Project description:In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.
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Project description:Colorectal cancer (CRC) is one of the most common cancers leading to high mortality. However, long-term administration of anti-tumor therapy for CRC is not feasible due to the side effects. Omega-3 polyunsaturated fatty acids (?-3 PUFAs), particularly DHA and EPA, exert protection against CRC, but the mechanisms are unclear. Here, we show that ?-3 PUFAs inhibit proliferation and induce apoptosis of CRC cells in vitro and alleviate AOM/DSS-induced mice colorectal cancer in vivo. Moreover, ?-3 PUFAs promote phosphorylation and cytoplasmic retention of YAP and this effect was mediated by MST1/2 and LATS1, suggesting that the canonical Hippo Pathway is involved in ?-3 PUFAs function. We further confirmed that increase of pYAP by ?-3 PUFAs was mediated by GPRs, including GPR40 and GPR120, which subsequently activate PKA via G?s, thus inducing the Hippo pathway activation. These data provide a novel DHA/EPA-GPR40/120-G?s-PKA-MST1/2-LATS1-YAP signaling pathway which is linked to ?-3 PUFAs-induced inhibition of cell proliferation and promotion of apoptosis in CRC cells, indicating a mechanism that could explain the anti-cancer action of ?-3 PUFAs.
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Project description:Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ. Experiment Overall Design: 16.7 billion nucleotides of shotgun, full length cDNA sequence data were generated using Illumina Genome Analyzer platforms with sequencing-by-synthesis (SBS) chemistry from 20 mRNA samples (Lister et al., 2008; Morin et al., 2008)(Mortazvi et al., 2008). mRNA samples were isolated post-mortem from the lateral hemispheres of the cerebellar cortices of 14 patients with SCZ and 6 control individuals (Paz et al., 2006; Bullock et al., In Press)(Table 1). Unrelated subjects were chosen to facilitate sampling of genetic heterogeneity (Freimer and Sabatti, 2004; McClellan et al., 2007). Cases and controls were approximately matched for age (cases, 45.2 + 11.8 years; controls 41.3 + 9.2 years), sex (all male), race, post-mortem interval (cases, 12.2 + 5.0 hours; controls 17.7 + 3.3 hours), cause of death, autopsy brain pH (cases, 6.54 + 0.19; controls 6.46 + 0.10) and RNA integrity number (cases, 8.06 + 0.53; controls, 7.87 + 0.41) (Table 1). 12.5 – 38.7 million, high quality sequences of length 32 – 36 bp were generated per sample (Table 2). Sequences were aligned to the human genome and RefSeq transcript databases using the algorithm GMAP, which allowed < 2 (< 6%) mismatches (Wu and Watanabe, 2005). There was little intra-sample variability in the number of sequences aligned to each locus from run-to-run or instrument-to-instrument (Fig. 1A; all source coefficient of variation 3.4%). 43.5 + 6.7% of sequences aligned to a transcript and 69.4 + 9.6% to the genome, evidence that annotation of mRNA isoforms in Homo sapiens is incomplete (Birney et al., 2007) (Table 2). 91% of alignments were unique (Table 2). Reads aligning to more than one location contained repetitive, paralogous, polymorphic or low complexity sequences and primarily mapped to untranslated regions or highly polymorphic gene families, such as major histocompatibility genes (Sugarbaker et al., 2008). Unmapped sequences did not align to mitochondrial or 1879 viral genomes, offering negative evidence of chronic viral etiology for SCZ in these patients.
2009-08-29 | E-GEOD-12297 | biostudies-arrayexpress
Project description:Mu-type opioid receptors are physiologically important G-protein-coupled receptors that are generally thought to recycle after agonist-induced endocytosis. Here we show that several alternatively spliced receptor variants fail to do so efficiently because of splice-mediated removal of an endocytic sorting sequence that is present specifically in the MOR1 variant. All of the recycling-impaired receptor variants were found to undergo proteolytic down-regulation more rapidly than MOR1, irrespective of moderate differences in endocytic rate, indicating that alternative splicing plays a specific role in distinguishing the trafficking itinerary of receptors after endocytosis. The recycling-impaired MOR1B variant was similar to MOR1 in its ability to mediate opioid-dependent inhibition of adenylyl cyclase, and to undergo opioid-induced desensitization in intact cells. Functional recovery (resensitization) of MOR1B-mediated cellular responsiveness after opioid removal, however, was significantly impaired (4-fold reduction in rate) compared with MOR1. To our knowledge the present results are the first to establish a role of alternative RNA processing in specifying the post-endocytic sorting of G-protein-coupled receptors between divergent and functionally distinct membrane pathways.
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Project description:G protein-coupled receptors with seven transmembrane alpha-helices (GPCRs) comprise the largest receptor superfamily and are involved in detecting a wide variety of extracellular stimuli. The availability of high-resolution crystal structures of five prototypical GPCRs, bovine and squid rhodopsin, engineered A(2A)-adenosine, beta(1)- and beta(2)-adrenergic receptors, permits comparative analysis of features common to these and likely all GPCRs. We provide an analysis of the distribution of water molecules in the transmembrane region of these GPCR structures and find conserved contacts with microdomains demonstrated to be involved in receptor activation. Colocalization of water molecules associating with highly conserved and functionally important residues in several of these GPCR crystal structures supports the notion that these waters are likely to be as important to proper receptor function as the conserved residues. Moreover, in the absence of large conformational changes in rhodopsin after photoactivation, we propose that ordered waters contribute to the functional plasticity needed to transmit activation signals from the retinal-binding pocket to the cytoplasmic face of rhodopsin and that fundamental features of the mechanism of activation, involving these conserved waters, are shared by many if not all family A receptors.
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Project description:Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal functions and have also been implicated in multiple neuropsychiatric disorders like fragile X syndrome, autism, and others. mGluR trafficking not only plays important roles in controlling the spatiotemporal localization of these receptors in the cell but also regulates the activity of these receptors. Despite this obvious significance, the cellular machineries that control the trafficking of group I metabotropic glutamate receptors in the central nervous system have not been studied in detail. The post-synaptic scaffolding protein tamalin has been shown to interact with group I mGluRs and also with many other proteins involved in protein trafficking in neurons. Using a molecular replacement approach in mouse hippocampal neurons, we show here that tamalin plays a critical role in the ligand-dependent internalization of mGluR1 and mGluR5, members of the group I mGluR family. Specifically, knockdown of endogenous tamalin inhibited the ligand-dependent internalization of these two receptors. Both N-terminal and C-terminal regions of tamalin played critical roles in mGluR1 endocytosis. Furthermore, we found that tamalin regulates mGluR1 internalization by interacting with S-SCAM, a protein that has been implicated in vesicular trafficking. Finally, we demonstrate that tamalin plays a critical role in mGluR-mediated internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, a process believed to be the cellular correlate for mGluR-dependent synaptic plasticity. Taken together, these findings reveal a mechanistic role of tamalin in the trafficking of group I mGluRs and suggest its physiological implications in the brain.
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