Emerging structural biology of lipid G protein‐coupled receptors

The first crystal structure of a G protein‐coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light‐induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high‐resolution structure of the adrenaline binding β₂‐adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high‐resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand‐binding and ligand‐mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid‐binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.     

Relevance of rhodopsin studies for GPCR activation

Rhodopsin, the dim-light photoreceptor present in the rod cells of the retina, is both a retinal-binding protein and a G protein-coupled receptor (GPCR). Due to this conjunction, it benefits from an arsenal of spectroscopy techniques that can be used for its characterization, while being a model system for the important family of Class A (also referred to as “rhodopsin-like”) GPCRs. For instance, rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007, it was the only GPCR for which a high-resolution crystal structure was available, so all structure–activity analyses on GPCRs, from structure-based drug discovery to studies of structural changes upon activation, were based on rhodopsin. At present, about a third of currently available GPCR structures are still from rhodopsin. In this review, I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First, the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region, which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second, a detailed analysis of the structure of the transmembrane domains in inactive, intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand-induced GPCR activation. Finally, the analysis of a conserved ligand-activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle, allows us to suggest that the structures of many of the currently available agonist-bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin, in other aspects rhodopsin is still at the forefront. For instance, the first studies of the structural basis of disease mutants in GPCRs, or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach, have been carried out in rhodopsin. Finally, due again to its unique properties among GPCRs, rhodopsin will likely play an important role in the application of X-ray free electron laser crystallography to time-resolved structural biology in membrane proteins. Rhodopsin, thus, still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.     

G 蛋白偶联受体寡聚体结构研究进展

G蛋白偶联受体(GPCR)是细胞膜上最大的一类受体,其通过构象变化激活下游G蛋白从而介导细胞响应多种来自内源和外界环境中的信号。自GPCR被发现以来,研究者就一直在努力解析GPCR的构象,x射线晶体衍射技术和GPCR蛋白质结晶技术的发展使得越来越多的GPCR单体在静息状态,以及与不同配体甚至G蛋白结合的晶体结构被成功解析。另一方面,FRET和电子显微技术的运用得到了GPCR二聚化和多聚化的多方面证据。本文将结合近年来该领域的进展,对GPCR寡聚体的结构和构象变化予以系统的综述,这些成果为研究GPCR的功能机制及其特异性的靶点药物开发提供了重要的基础。     

Nonenzymatic rapid control of GIRK channel function by a G protein-coupled receptor kinase

G protein-coupled receptors (GPCRs) respond to agonists to activate downstream enzymatic pathways or to gate ion channel function. Turning off GPCR signaling is known to involve phosphorylation of the GPCR by GPCR kinases (GRKs) to initiate their internalization. The process, however, is relatively slow and cannot account for the faster desensitization responses required to regulate channel gating. Here, we show that GRKs enable rapid desensitization of the G protein-coupled potassium channel (GIRK/Kir3.x) through a mechanism independent of their kinase activity. On GPCR activation, GRKs translocate to the membrane and quench channel activation by competitively binding and titrating G protein βγ subunits away from the channel. Of interest, the ability of GRKs to effect this rapid desensitization depends on the receptor type. The findings thus reveal a stimulus-specific, phosphorylation-independent mechanism for rapidly downregulating GPCR activity at the effector level.     

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.     

Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D₃ dopamine receptor (D₃R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D₃R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.     

G protein activation by serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient

The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and β(2)-adrenergic receptors, activate G proteins. By using wild type and mutant serotonin type 4 receptors (5-HT(4)Rs) (including a 5-HT(4)-RASSL) expressed in COS-7 cells as models of class A GPCRs, we show that activation of one protomer in a dimer was sufficient to stimulate G proteins. However, coupling efficiency was 2 times higher when both protomers were activated. Expression of combinations of 5-HT(4), in which both protomers were able to bind to agonists but only one could couple to G proteins, suggested that upon agonist occupancy, protomers did not independently couple to G proteins but rather that only one G protein was activated. Coupling of a single heterotrimeric G(s) protein to a receptor dimer was further confirmed in vitro, using the purified recombinant WT RASSL 5-HT(4)R obligatory heterodimer. These results, together with previous findings, demonstrate that, differently from class C GPCR dimers, class A GPCR dimers have pleiotropic activation mechanisms.     

The structure of active opsin as a basis for identification of GPCR agonists by dynamic homology modelling and virtual screening assays

Most of the currently available G protein-coupled receptor (GPCR) crystal structures represent an inactive receptor state, which has been considered to be suitable only for the discovery of antagonists and inverse agonists in structure-based computational ligand screening. Using the β(2)-adrenergic receptor (B2AR) as a model system, we show that a dynamic homology model based on an "active" opsin structure without further incorporation of experimental data performs better than the crystal structure of the inactive B2AR in finding agonists over antagonists/inverse agonists. Such "active-like state" dynamic homology models can therefore be used to selectively identify GPCR agonists in in silico ligand libraries.     

Cis- and trans-activation of hormone receptors: the LH receptor

G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.     

G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction

The mechanism by which G protein-coupled receptors (GPCRs) translate extracellular signals into cellular changes initially was envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein into its alpha and betagamma subunits, both of which can activate or inhibit various downstream effector molecules. The plethora of recently described multidomain scaffolding proteins and accessory/chaperone molecules that interact with GPCR, including GPCR themselves as homo- or heterodimers, provides for diverse molecular mechanisms for ligand recognition, signalling specificity, and receptor trafficking. This review will summarize the recently described GPCR-interacting proteins and their individual functional roles, as understood. Implicit in the search for the functional relevance of these interactions is the expectation that enhancement or disruption of target cell-specific events could serve as highly selective therapeutic opportunities.     

A conformational trigger for activation of a G protein by a G protein-coupled receptor

G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest. Three-dimensional structures of the ground state of only one GPCR, rhodopsin, are currently available, but since the G protein cannot bind to this structure, these structures did not lead to an understanding of the activation process. The recent publication of an excited state structure for the same GPCR (and comparison to the ground state structures), in conjunction with other recent biochemical data, provides new insight into G protein activation. We find that the structure data and the biochemical data, for the first time, point to a specific mode of interaction between the G protein and the receptor. Furthermore, we find that transducin (G(t)) must alter its conformation to bind to the activated receptor; the "lock and key" fit heretofore expected is likely not the correct model. We suggest that a conformational distortion, driven by the energy of binding, is induced in G(t) when it binds to the activated receptor. The conformational change in turn enables the exchange of GTP for GDP and the dissociation of the subunits. This is an example of "induced fit" originally proposed by Koshland to describe enzyme-substrate interactions.     

Opioid receptor random mutagenesis reveals a mechanism for G protein-coupled receptor activation

The high resolution structure of rhodopsin has greatly enhanced current understanding of G protein-coupled receptor (GPCR) structure in the off-state, but the activation process remains to be clarified. We investigated molecular mechanisms of delta-opioid receptor activation without a preconceived structural hypothesis. Using random mutagenesis of the entire receptor, we identified 30 activating point mutations. Three-dimensional modeling revealed an activation path originating from the third extracellular loop and propagating through tightly packed helices III, VI and VII down to a VI-VII cytoplasmic switch. N- and C-terminal determinants also influence receptor activity. Findings for this therapeutically important receptor may apply to other GPCRs that respond to diffusible ligands.     

G protein-mediated stretch reception

Mechanosensation and -transduction are important for physiological processes like the senses of touch, hearing, and balance. The mechanisms underlying the translation of mechanical stimuli into biochemical information by activating various signaling pathways play a fundamental role in physiology and pathophysiology but are only poorly understood. Recently, G protein-coupled receptors (GPCRs), which are essential for the conversion of light, olfactory and gustatory stimuli, as well as of primary messengers like hormones and neurotransmitters into cellular signals and which play distinct roles in inflammation, cell growth, and differentiation, have emerged as potential mechanosensors. The first candidate for a mechanosensitive GPCR was the angiotensin-II type-1 (AT(1)) receptor. Agonist-independent mechanical receptor activation of AT(1) receptors induces an active receptor conformation that appears to differ from agonist-induced receptor conformations and entails the activation of G proteins. Mechanically induced AT(1) receptor activation plays an important role for myogenic vasoconstriction and for the initiation of cardiac hypertrophy. A growing body of evidence suggests that other GPCRs are involved in mechanosensation as well. These findings highlight physiologically relevant, ligand-independent functions of GPCRs and add yet another facet to the polymodal activation spectrum of this ubiquitous protein family.     

Lipidic cubic phase technologies for membrane protein structural studies

Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

Multiple activation steps of the N-formyl peptide receptor

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

Chemokine receptor CCR5: insights into structure, function, and regulation

CC chemokine receptor 5 (CCR5) is a seven-transmembrane, G protein-coupled receptor (GPCR) which regulates trafficking and effector functions of memory/effector T-lymphocytes, macrophages, and immature dendritic cells. It also serves as the main coreceptor for the entry of R5 strains of human immunodeficiency virus (HIV-1, HIV-2). Chemokine binding to CCR5 leads to cellular activation through pertussis toxin-sensitive heterotrimeric G proteins as well as G protein-independent signalling pathways. Like many other GPCR, CCR5 is regulated by agonist-dependent processes which involve G protein coupled receptor kinase (GRK)-dependent phosphorylation, beta-arrestin-mediated desensitization and internalization. This review discusses recent advances in the elucidation of the structure and function of CCR5, as well as the complex mechanisms that regulate CCR5 signalling and cell surface expression.