Identification of G protein-coupled receptor genes from the human genome sequence

We have identified novel G protein-coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands.     

昆虫非典型嗅觉受体Orco的功能和分子结构研究进展

嗅觉受体是参与昆虫嗅觉识别过程的一类重要蛋白。在昆虫的众多嗅觉受体中, 有一类受体明显不同于其他受体, 被称为Orco。该受体基因在不同昆虫种间高度保守, 且表达广泛。Orco在昆虫嗅觉识别过程中发挥关键作用。采用基因突变或RNAi等技术使Orco基因沉默后, 昆虫会出现严重的嗅觉缺陷, 但Orco本身不与气味配体结合, 它与传统嗅觉受体形成复合体Or-Orco, 促进传统嗅觉受体在神经元树突膜上的定位并维持其稳定性, 提高传统嗅觉受体对气味反应的效率。昆虫嗅觉受体的结构与脊椎动物的G蛋白偶联受体相似, 均有7个跨膜区, 但二者的膜拓扑结构相反, 昆虫嗅觉受体的N末端位于细胞质膜内, C末端在细胞质膜外, Orco与传统嗅觉受体通过保守的C末端区域相互作用形成一种新型的配体门控离子通道--Or-Orco复合体。阐明Orco在昆虫嗅觉识别中的功能机制, 可为开创基于昆虫嗅觉行为干扰的新的害虫防治措施提供基础。     

Amphioxus (Branchiostoma floridae) has orthologs of vertebrate odorant receptors

Background  

A common feature of chemosensory systems is the involvement of G protein-coupled receptors (GPCRs) in the detection of environmental stimuli. Several lineages of GPCRs are involved in vertebrate olfaction, including trace amine-associated receptors, type 1 and 2 vomeronasal receptors and odorant receptors (ORs). Gene duplication and gene loss in different vertebrate lineages have lead to an enormous amount of variation in OR gene repertoire among species; some fish have fewer than 100 OR genes, while some mammals possess more than 1000. Fascinating features of the vertebrate olfactory system include allelic exclusion, where each olfactory neuron expresses only a single OR gene, and axonal guidance where neurons expressing the same receptor project axons to common glomerulae. By identifying homologous ORs in vertebrate and in non-vertebrate chordates, we hope to expose ancestral features of the chordate olfactory system that will help us to better understand the evolution of the receptors themselves and of the cellular components of the olfactory system.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

Characteristic amino acid combinations in olfactory G protein-coupled receptors

The human olfactory subgenome has recently been fully characterized with over 1000 genes. Although as many as two thirds of them are expected to be pseudogenes, it still leaves us with about half of all human G protein-coupled receptors being olfactory. It is therefore of great interest to characterize olfactory receptors with high precision. Usually it is done through sequence motifs that are not fully conserved, making an exact characterization difficult. In this paper, we propose a rule-based characterization of olfactory receptors derived from a multiple sequence alignment of human GPCRs. We show that just seven alignment sites are sufficient to characterize 99% of human olfactory GPCRs with one feature, a tyrosine at site 7.41, being of particular importance. We also show dependencies between sites near the extracellular and intracellular region of a membrane-embedded receptor, indicating that olfactory receptors are characterized by a combination of important residues in these two areas, whereas nonolfactory receptors tend to have residues of lower importance at the same sites.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

A machine learning based method for the prediction of G protein-coupled receptor-binding PDZ domain proteins

G protein-coupled receptors (GPCRs) are part of multi-protein networks called ‘receptosomes’. These GPCR interacting proteins (GIPs) in the receptosomes control the targeting, trafficking and signaling of GPCRs. PDZ domain proteins constitute the largest protein family among the GIPs, and the predominant function of the PDZ domain proteins is to assemble signaling pathway components into close proximity by recognition of the last four C-terminal amino acids of GPCRs. We present here a machine learning based approach for the identification of GPCR-binding PDZ domain proteins. In order to characterize the network of interactions between amino acid residues that contribute to the stability of the PDZ domain-ligand complex and to encode the complex into a feature vector, amino acid contact matrices and physicochemical distance matrix were constructed and adopted. This novel machine learning based method displayed high performance for the identification of PDZ domain-ligand interactions and allowed the identification of novel GPCR-PDZ domain protein interactions.     

Evolution of odorant receptors

Odorant receptors (ORs) located in the nasal epithelium, at the ciliated surface of olfactory sensory neurons, represent the initial step of a transduction cascade that leads to odor detection. ORs form the largest and most diverse family of G-protein-coupled receptors (GPCRs). They are encoded by a multigene family that has been partially characterized in cyclostomes, teleosts, amphibia, birds and mammals, as well as in Drosophila melanogaster and the nematode Caenorhabditis elegans. As new sequence data emerge, it is increasingly clear that OR primary structure can vary dramatically across phyla. Some chemoreceptors are encoded by genes with little sequence similarity to the prototypical ORs originally isolated in mammals. A large number of sequences are now available allowing a detailed study of the evolutionary implications of OR diversity across species. This review discusses the evolutionary implications of the divergent primary structures of chemoreceptors with identical functions.     

Role of Ubiquitination in Endocytic Trafficking of G‐Protein‐Coupled Receptors

Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G‐protein‐coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin‐dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin‐dependent and ‐independent regulation that may be specific to animal cells.     

强啡肽单抗融合蛋白的克隆、表达及功能分析

G蛋白偶联受体(GPCR)长期以来是最重要的药物靶点家族,小分子药物层出不穷。然而受研发难度的限制,针对GPCR的抗体或大分子类药物屈指可数。我们利用选择性靶向κ阿片受体(KOR)且无法激活下游信号的单克隆抗体连接强啡肽(Dynorphin)基因、HEK 293F系统进行表达,纯化获得KOR强啡肽单抗融合蛋白,我们将此融合蛋白命名为APF(antibody-peptide fusion)。结果显示,获得的单抗融合蛋白二级结构未显著改变,保持了Dynorphin活性,可激活KOR相关下游蛋白(Gi)活性,调动β-arrestin信号。结果证明,基因层面实现抗体药物改构的可行性,该法可指导新一代抗体偶联药物的改造,为以GPCR为靶点的大分子药物开发提供了新的空间。     

The G protein-coupled receptor subset of the chicken genome

G protein-coupled receptors (GPCRs) are one of the largest families of proteins, and here we scan the recently sequenced chicken genome for GPCRs. We use a homology-based approach, utilizing comparisons with all human GPCRs, to detect and verify chicken GPCRs from translated genomic alignments and Genscan predictions. We present 557 manually curated sequences for GPCRs from the chicken genome, of which 455 were previously not annotated. More than 60% of the chicken Genscan gene predictions with a human ortholog needed curation, which drastically changed the average percentage identity between the human-chicken orthologous pairs (from 56.3% to 72.9%). Of the non-olfactory chicken GPCRs, 79% had a one-to-one orthologous relationship to a human GPCR. The Frizzled, Secretin, and subgroups of the Rhodopsin families have high proportions of orthologous pairs, although the percentage of amino acid identity varies. Other groups show large differences, such as the Adhesion family and GPCRs that bind exogenous ligands. The chicken has only three bitter Taste 2 receptors, and it also lacks an ortholog to human TAS1R2 (one of three GPCRs in the human genome in the Taste 1 receptor family [TAS1R]), implying that the chicken's ability and mode of detecting both bitter and sweet taste may differ from the human's. The chicken genome contains at least 229 olfactory receptors, and the majority of these (218) originate from a chicken-specific expansion. To our knowledge, this dataset of chicken GPCRs is the largest curated dataset from a single gene family from a non-mammalian vertebrate. Both the updated human GPCR dataset, as well the chicken GPCR dataset, are available for download.     

Mechanisms of selective G protein–coupled receptor localization and trafficking

The trafficking of G protein–coupled receptors (GPCRs) to different membrane compartments has recently emerged as being a critical determinant of the signaling profiles of activation. GPCRs, which share many structural and functional similarities, also share many mechanisms that traffic them between compartments. This sharing raises the question of how the trafficking of individual GPCRs is selectively regulated. Here, we will discuss recent studies addressing the mechanisms that contribute to selectivity in endocytic and biosynthetic trafficking of GPCRs.     

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.     

CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum

The molecular mechanisms regulating G protein‐coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer‐based proteomic screen, we identified a novel GPCR‐interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER‐resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the β2‐adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co‐immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo‐sorting receptor, recruiting GPCRs into COPII vesicles .      

Therapeutic antibodies directed at G protein-coupled receptors

G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in over-expressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.     

The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks

All cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

Background

Dysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.

Methodology/Principal Findings

We used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.

Conclusions/Significance

These results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis.     

FMRFamide related peptide ligands activate the Caenorhabditis elegans orphan GPCR Y59H11AL.1

G-protein coupled receptors (GPCRs) are ancient molecules that can sense environmental and physiological signals. Currently, the majority of the predicted Caenorhabditis elegans GPCRs are orphan. Here, we describe the characterization of such an orphan C. elegans GPCR, which is categorized in the tachykinin-like group of receptors. Since the C. elegans genome predicts only one tachykinin-like peptide (SFDRMGGTEFGLM), which could not activate the receptor, we hypothesized that one or some of the numerous FMRFamide related peptides (FaRPs) could be the cognate ligands for this receptor. This hypothesis was based on the suggestion that RFamides may be ancestral neuropeptides, from which a lot of the amidated neuropeptides, including tachykinins, derived. Indeed, we found that the orphan receptor encoded by the Y59H11AL.1 gene is activated by several C. elegans neuropeptides, including SPMERSAMVRFamide. These peptides activate the receptor in a concentration-dependent way.     

D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.