Diversity and Modularity of G Protein-Coupled Receptor Structures

Article · Literature Review · January 2012with131 Reads
DOI: 10.1016/j.tips.2011.09.003 · Source: PubMed
G protein-coupled receptors (GPCRs) comprise the most 'prolific' family of cell membrane proteins, which share a general mechanism of signal transduction, but greatly vary in ligand recognition and function. Crystal structures are now available for rhodopsin, adrenergic, and adenosine receptors in both inactive and activated forms, as well as for chemokine, dopamine, and histamine receptors in inactive conformations. Here we review common structural features, outline the scope of structural diversity of GPCRs at different levels of homology, and briefly discuss the impact of the structures on drug discovery. Given the current set of GPCR crystal structures, a distinct modularity is now being observed between the extracellular (ligand-binding) and intracellular (signaling) regions. The rapidly expanding repertoire of GPCR structures provides a solid framework for experimental and molecular modeling studies, and helps to chart a roadmap for comprehensive structural coverage of the whole superfamily and an understanding of GPCR biological and therapeutic mechanisms.
    • The peak with molecular weight 10.5 represents THM67H8-methionine. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), n-dodecylphosphocholine (DPC), and dimyristoylphosphocholine/dihexanoylphosphocholine (DMPC/DHPC) isotropic bicelles (q = 0.16).
    [Show abstract] [Hide abstract] ABSTRACT: This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly ¹⁵N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 0.42 resulted in peak crowding. These studies demonstrate that GPCRs folding especially in bicelles is protein-specific and requires the right mix of the longer chain and shorter chain lipids to provide the right environment for proper folding. These findings will allow further development of novel membrane mimetics to provide greater diversity of lipid mixtures than those currently being employed for GPCR stability and folding, which are critical for both X-ray and NMR studies of GPCRs.
    Full-text · Article · Feb 2017
    • The intracellular domains of GPCRs are found to be conserved with the exception of intracellular loop 3, which is the most variable region of GPCRs [63]. The intracellular region is involved in several facets of GPCR signaling where residues in the cytoplasmic ends of TM helices, intracellular loops, and C-terminus bind to various downstream signaling molecules such as G proteins, GPCR kinases, and arrestins [64, 65]. Additionally, the length of intracellular loops vary in GPCRs, especially ICL3, which is also known to be innately disordered [66] .
    [Show abstract] [Hide abstract] ABSTRACT: Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.
    Article · Dec 2016
    • Additionally, other experiments also hint to functional dimers of these receptors (Angers et al., 2000; Jastrzebska et al., 2015). In general, the ongoing development and improvement of membrane protein crystallography allowed to resolve several crystal structures of GPCRs (Ghosh et al., 2015), frequently with the receptors in stable dimeric conformations (Rosenbaum et al., 2009; Katritsch et al., 2012). However, crystal dimer configurations may not necessarily reflect native dimer configurations, since the crystallization may require sequence modifications and in particular since the crystal environment differs considerably from the membrane environment in vivo.
    [Show abstract] [Hide abstract] ABSTRACT: The dimerization or even oligomerization of G protein coupled receptors (GPCRs) causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signaling complexes. Recent findings further suggest that the surrounding membrane, i.e., its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.
    Full-text · Article · Oct 2016
    • Small-molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand-binding site. Some of these ligands achieve selectivity for a subset of tissue-or pathwayspecific signaling outcomes, which is called selective modulation, functional selectivity, or biased signaling, through structural mechanisms that are poorly understood (Frolik et al, 1996; Nettles & Greene, 2005; Overington et al, 2006; Katritch et al, 2012; Wisler et al, 2014). For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex â ; AstraZeneca) or raloxifene (Evista â ; Eli Lilly) (Fig 1A)block the ERa-mediated proliferative effects of the native estrogen, 17b-estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003).
    [Show abstract] [Hide abstract] ABSTRACT: Some estrogen receptor-α (ERα)-targeted breast cancer therapies such as tamoxifen have tissue-selective or cell-specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell-specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X-ray crystallography. Ligands that regulate the dynamics and stability of the coactivator-binding site in the C-terminal ligand-binding domain, called activation function-2 (AF-2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter-atomic distance in the ligand-binding domain predicted their proliferative effects. In contrast, the N-terminal coactivator-binding site, activation function-1 (AF-1), determined cell-specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.
    Full-text · Article · Apr 2016
    • The recent crystal and NMR structures of chemokine-CKR complexes provide clues that far from following a two-site convention, interactions are diverse and highly specific for each individual chemokine-CKR pair at the extracellular surface [8, 9, 13]. Similarly, a surfeit of 7TMR crystal structures over the past decade is defining how the conserved TM architecture recognizes diverse ligand types and triggers unique signaling outcomes [58, 59]. In particular, the early 7TMR crystal structures divided the orthosteric-binding site (i.e. the 'main' ligand binding pocket) into two subpockets [58,[60][61][62]: the major subpocket consists of the cavity defined by TMs 4, 5, and 6, and the minor subpocket by TMs 1 and 2. TMs 3 and 7 occupy the interface between the two subpockets and stabilize ligand-CKR interactions in either subpocket [3, 58, 63, 64].
    [Show abstract] [Hide abstract] ABSTRACT: Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions.
    Article · Apr 2016
    • However, for the non-olfactory receptors, either ECL1 or ECL2 is longer than 20 amino acids, or both loops are shorter than 20 amino acids. This observation is distinct from the trend of GPCRs in general, of which the 7TM helical bundle has been the most conserved component (Katritch et al., 2012), across the over 400 various odorant receptors (Jiang and Matsunami, 2015), the most conserved domains are the intracellular loops and the seventh transmembrane helix (helix VII), while the sequence diversity of helices III, IV, and V to which the odorant molecules bind is very high (Gao et al., 2010; de March et al., 2015). These two characteristics may contribute to the low expression level and instability of the olfactory receptors.
    [Show abstract] [Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0263-8) contains supplementary material, which is available to authorized users.
    Full-text · Article · Apr 2016
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