Interaction of class A G protein-coupled receptors with G proteins

A model for interaction of classA G protein-coupled receptor with the G protein G(alpha) subunit is proposed using the rhodopsin-transducin (RD/Gt) prototype. The model combines the resolved interactions/distances, essential in the active RD*/Gt system, with the structure of Gt(alpha) C-terminal peptide bound to RD* while stabilizing it. Assuming the interactions involve conserved parts of the partners, the model specifies the conserved Helix 2 non-polar X- - -X, Helix 3 DRY and Helix 7/8 NP- -Y- - F RD* motifs interacting with the Gt(alpha) C-terminal peptide, in compliance with the structure of the latter. A concomitant role of Gt(alpha) and Gt(gamma) C-termini in stabilizing RD* could possibly be resolved assuming a receptor dimer as requisite for G protein activation.     

Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

Lysophospholipid G protein-coupled receptors

The many biological responses documented for lysophospholipids that include lysophosphatidic acid and sphingosine 1-phosphate can be mechanistically attributed to signaling through specific G protein-coupled receptors. At least nine receptors have now been identified, and the total number is likely to be larger. In this brief review, we note cogent features of lysophospholipid receptors, including the current nomenclature, signaling properties, development of agonists and antagonists, and physiological functions.     

A low resolution model for the interaction of G proteins with G protein-coupled receptors

A model is presented for the interaction between G proteins and G protein-coupled receptors. The model is based on the fact that this interaction shows little specificity and thus conserved parts of the G proteins have to interact with conserved parts of the receptors. These parts are a conserved negative residue in the G protein, a fully conserved arginine in the receptor and a series of residues that are not conserved but always hydrophobic like the hydrophobic side of the C-terminal helix of the G protein and the hydrophobic side of a helix in the C-terminal domain of the receptor. Other, mainly cytosolic, factors determine the specificity and regulation of this interaction. The relation between binding and activation will be shown. A large body of experimental evidence supports this model. Despite the fact that the model does not provide atomic resolution, it can be used to explain some experimental data that would otherwise seem inexplicable, and it suggests experiments for its falsification or verification.     

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.     

G protein-coupled receptors in energy homeostasis

G-protein coupled receptors (GPCRs) compromise the largest membrane protein superfamily which play vital roles in physiological and pathophysiological processes including energy homeostasis. Moreover, they also represent the up-to-date most successful drug target. The gut hormone GPCRs, such as glucagon receptor and GLP-1 receptor, have been intensively studied for their roles in metabolism and respective drugs have developed for the treatment of metabolic diseases such as type 2 diabetes (T2D). Along with the advances of biomedical research, more GPCRs have been found to play important roles in the regulation of energy homeostasis from nutrient sensing, appetite control to glucose and fatty acid metabolism with various mechanisms. The investigation of their biological functions will not only improve our understanding of how our body keeps the balance of energy intake and expenditure, but also highlight the possible drug targets for the treatment of metabolic diseases. The present review summarizes GPCRs involved in the energy control with special emphasis on their pathophysiological roles in metabolic diseases and hopefully triggers more intensive and systematic investigations in the field so that a comprehensive network control of energy homeostasis will be revealed, and better drugs will be developed in the foreseeable future.     

Selectivity in the oligomerisation of G protein-coupled receptors

G protein-coupled receptors can exist as dimers and/or higher order oligomers. Such quaternary structure appears central to their plasma membrane delivery and, potentially, to function. Recent evidence that these receptors can form hetero- as well as homo-dimers/oligomers has significant implications for pharmacology and pathophysiology. Knowledge of the basis and selectivity of GPCR hetero-dimerisation is thus vital. Current understanding of these areas is reviewed.     

Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors

We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha16. We now show that, compared with Galpha16, the use of chimeric Galphaq subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect Gi-coupled GPCRs into a Gq-type signal transduction pathway. We coexpressed human Gi-coupled GPCRs, G protein alpha subunits (either a chimeric Galphaq or Galpha16), and the calcium-sensitive reporter protein aequorin in Sf9 cells using a nonlytic protein expression system, and measured agonist-induced intracellular calcium flux using a luminometer. Three of the GPCRs (serotonin 1A, 1D, and dopamine D2) were functionally redirected into a Gq-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha16. We determined agonist concentration-response relationships for all three receptors, which yielded EC50 values comparable with those achieved in mammalian cell-based assay systems. However, three other Gi-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha16. Possible reasons and solutions for this result are discussed.     

Mechanisms of G protein-coupled receptors regulation

Within the last two decades of studies in the ever-expanding field of GPCR signaling, challenging insights were adopted. Growing evidence now asists the shift from classical linear model of signaling towards a considerably complex network of signaling pathways with many shared proteins and cross-talks. Considering the extensive and intriguing network of pathways activated by these receptors, it is apparent that multi-level system of regulation must exist to rigorously modulate the amplitude, duration and spatial aspects of the GPCR signaling. This review summarizes the principal mechanisms of GPCR regulation and gives the overview of recent advances in this field of research.     

Structural biology of G protein-coupled receptors

More than 60% of the current drugs are based on G protein-coupled receptors. Paradoxically, high-resolution structures are not available to facilitate rational drug design. Difficulties in expression, purification, and crystallization of these transmembrane receptors are the reasons for the low success rate. Recent individual and network-based technology development has significantly improved our knowledge of structural biology and might soon bring a major breakthrough in this area.     

Agonist-bound structures of G protein-coupled receptors

G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the β(2)-adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.     

Splice variants of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are encoded by a vast gene superfamily, reflecting the large number of ligands that must be specifically recognized at any given cell surface. The discovery that the variety of GPCRs is further expanded through the generation of splice variants was therefore somewhat surprising. Studies of the functional consequences of alternative splicing have focused on ligand binding, signaling, constitutive activity, and downregulation. However, GPCRs also appear to interact directly with many other intracellular proteins in addition to G proteins. Intriguingly, the domains involved in these interactions are the predominant sites of variation arising through splicing.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.     

G蛋白偶联受体的结构生物学研究

G蛋白偶联受体(G protein-coupled receptor,GPCR)在细胞信号转导过程中发挥关键的生理学功能,是极其重要的药物靶标,其三维结构信息对功能研究以及新药研发具有十分重要的意义。近年来,新技术的发展和应用使GPCR的结构生物学研究发生了跨越式的发展,本文简要回顾这些新的技术和方法以及已解析的GPCR三维结构,并以CCR5和P2Y12R两种受体的结构为例来具体阐明现阶段GPCR结构生物学研究的内容和意义。     

Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins

The histamine H1 receptor and the alpha1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([35S]thio)triphosphate ([35S]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [35S]GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.     

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.     

A GAIN in understanding autoproteolytic G protein-coupled receptors and polycystic kidney disease proteins

EMBO J 31 6, 1364–1378 (2012); published online January142012A large and poorly understood class of G protein-coupled receptors (GPCRs) are involved in cell adhesion and contain an autoproteolytic site known as the GPCR proteolysis site (GPS) located immediately N-terminal to the first transmembrane span. This motif of ∼50 amino acids is also found juxtaposed to the first transmembrane span of an unrelated family of proteins associated with polycystic kidney disease (PKD), but its structural and functional roles were not clear. In this issue of The EMBO Journal, Arac et al use X-ray crystallography to show that the GPS motif is merely the C-terminal end of a much larger GPCR autoproteolysis inducing (GAIN) domain. Atomic models for two of these ancient domains allow one to map the sites of mutations associated with cancer or PKD, and hint at functional roles other than autoproteolysis.Autoproteolysis occurs in a wide variety of proteins where it typically leads to an essential structural reorganization or the release of an activated fragment. One such autoproteolytic site, called the GPS motif, is found in cell-adhesion GPCRs (Ichtchenko et al, 1999). Like other GPCRs, cell-adhesion GPCRs contain a heptahelical transmembrane domain that is expected to couple with heterotrimeric G proteins inside the cell. However, they also have unusually complex and diverse extracellular regions that contain domains homologous to those typically involved in cell adhesion (Yona et al, 2008).The extracellular regions of cell-adhesion GPCRs all contain a conserved stalk region that ends in the GPS motif, which is cleaved in the endoplasmic reticulum just after protein synthesis. Araç et al (2012) crystallized the stalk region (∼320 amino acids) along with the preceding HormR domain (∼70 amino acids) of two different cell-adhesion GPCRs: latrophilin (also called CIRL1 or CL1) and brain angiogenesis inhibitor 3 (BAI3). The resulting structures demonstrate that the stalk and GPS motif together form a large domain with α-helical and β-sandwich subdomains (Figure 1). The site of autoproteolysis, defined by the consensus sequence HL↓(T/S), occurs in a tight turn between the last two β strands of the domain. Because the entire domain was required for autoproteolysis, the authors renamed the stalk and GPS motif the GAIN domain. Sequence analysis supports the existence of a similar domain in all cell-adhesion GPCRs and homologues of polycystic kidney disease 1 (PKD1 or polycystin-1).Open in a separate windowFigure 1The conserved structural core of the extracellular regions of cell-adhesion GPCRs, as revealed by new structures of latrophilin and BAI3. Arac et al revealed that regions formerly known as the stalk and the GPCR autoproteolyic site (GPS) fold into a single GAIN domain that is conserved in all 33 mammalian cell-adhesion GPCRs and in proteins related to PKD1. The GAIN domain catalyses its own proteolysis in a tight turn between the last two strands of the domain. The α-helical subdomain of the GAIN domains of latrophilin and BAI3 interacts with a HormR domain, although this domain is not found in all cell-adhesion GPCRs. However, this interdomain contact, and/or a direct interaction with the transmembrane helical bundle, may allow the GAIN domain to autoinhibit transmembrane signalling to heterotrimeric G proteins or other factors inside the cell.For reasons that are not clear, the latrophilin GAIN domain used in this study was cleaved, whereas that of BAI3 was not. However, this allowed Araç et al to compare structures of wild-type GAIN domains before and after autoproteolysis. Before cleavage, the scissile bond exists in a strained conformation, and the position of adjacent catalytic residues is consistent with an Ntn hydrolase mechanism (Brannigan et al, 1995; Lin et al, 2004). After cleavage, the cleaved fragment (the β13 strand) appears firmly anchored within the GAIN domain by extensive backbone hydrogen bonds and hydrophobic interactions.But what is the functional role of autoproteolysis? Early work suggested that it may be required for efficient membrane transport because mutation of residues in the GPS motif were known to impair proper membrane trafficking (Krasnoperov et al, 2002). However, other studies, including that of Araç et al, have shown that this is not always the case (Qian et al, 2002). More likely, mutations in the core of the GAIN domain lead to protein folding defects, which would in turn impair trafficking (Lin et al, 2004). Autoproteolysis may therefore represent a mechanism by which the GAIN domain is locked into its functional and presumably more stable state after proper folding occurs. For this reason, it would be interesting to determine the relative thermostability of a GAIN domain before and after cleavage. Still, there remains evidence that GAIN domain autocleavage has functional consequences. Mice that express non-cleavable PKD1 exhibit abnormal kidney development (Yu et al, 2007). Comparison of the latrophilin and BAI3 GAIN domain structures also reveals a subtle conformational change in the β13 strand after cleavage that could, in principle, impact the ability of the domain to interact with other signalling domains. Finally, it has been reported that the cleaved extracellular region of latrophilin is dissociable from its transmembrane domain, and that ligand-induced reassociation may play a role in signalling (Silva and Ushkaryov, 2010).The GAIN domains of latrophilin and BAI homologues are mutated in human cancer, whereas the PKD1 GAIN domain is frequently mutated in autosomal dominant PKD (ADPKD). Most of the cancer-associated mutations map to the surface of the GAIN domain and did not affect autoproteolysis, implying that they disrupt the intermolecular contacts of the domain. In support of this idea, the authors showed that the latrophilin GAIN domain is the binding site for the black widow toxin α-latrotoxin. In contrast, most of the ADPKD mutations in the PKD1 GAIN domain interfered with autoproteolysis and/or protein folding, hinting at a different role for the domain in PKD1-related proteins. Furthermore, the GAIN domains of latrophilin and BAI3 interact with the preceding HormR domain in a manner that would block the interaction of homologous HormR domains with hormones. The GAIN domain may therefore serve to autoinhibit signalling activity, either by blocking the interactions of other extracellular domains or, in the case of cell-adhesion GPCRs, by modulating the activity of the adjacent seven transmembrane bundle (Figure 1). Indeed, truncation of the N-terminus of GPR56, including the bulk of the GAIN domain, leads to constitutive activation (Paavola et al, 2011). Thus, the structural stage is now set for studies aimed at understanding the molecular basis for signal transduction through cell-adhesion GPCRs and PKD1 proteins, and how defects in these proteins lead to disease.