GPCR dimerisation

The concept that GPCRs exist and potentially function as dimers and/or higher oligomers has progressed recently from hypothesis to being widely accepted. A range of techniques has contributed to this understanding, including co-immunoprecipitation and various forms of fluorescence and bioluminescence resonance energy transfer. Although co-immunoprecipitation studies indicate the capacity of a wide range of GPCRs to form hetero-dimers as well as homo-dimers, this approach is not well suited to examine selectivity of interactions. Both bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) have been applied to the detection of GPCR dimerisation in intact cells and BRET and FRET have been used to attempt to quantitate the fraction of GPCRs present as dimers. Following heterologous expression, a considerable fraction of many GPCRs is not fully processed and is trafficked to the proteasome or lysosome for destruction. A distinct limitation of both BRET and conventional FRET approaches is that both the energy donor and energy acceptor tags are inside the cell. Time-resolved FRET employing N-terminally epitope-tagged GPCRs has been used to allow detection only of dimers trafficked successfully to the cell surface. Reports indicating the appearance of distinct pharmacology and function following co-expression of two GPCRs are fascinating. Much remains to be examined, however, on the specificity and mechanisms of these interactions and to develop techniques to monitor the function only of hetero-dimers when the corresponding homo-dimers must also be present.     

GPCR homo-oligomerization

Cartoon depictions of (a) Dynamic interchange between receptor monomers, (homo, orange GPCR1, and hetero, orange GPCR1 and green GPCR2) dimers and oligomers which may be ligand regulated (orange sphere). (b) Signalling cross-talk between protomers of a dimer. Left: ligand (orange sphere) binds to one protomer and the conformational change transmitted to the second protomer results in signalling through activation of a Gα subunit (blue). Right: ligand (green sphere) binding to the second protomer may modulate signalling (large or small yellow flash).

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S-nitrosylation-regulated GPCR signaling

G protein-coupled receptors (GPCRs) are the most numerous and diverse type of cell surface receptors, accounting for about 1% of the entire human genome and relaying signals from a variety of extracellular stimuli that range from lipid and peptide growth factors to ions and sensory inputs. Activated GPCRs regulate a multitude of target cell functions, including intermediary metabolism, growth and differentiation, and migration and invasion. The GPCRs contain a characteristic 7-transmembrane domain topology and their activation promotes complex formation with a variety of intracellular partner proteins, which form basis for initiation of distinct signaling networks as well as dictate fate of the receptor itself. Both termination of active GPCR signaling and removal from the plasma membrane are controlled by protein post-translational modifications of the receptor itself and its interacting partners. Phosphorylation, acylation and ubiquitination are the most studied post-translational modifications involved in GPCR signal transduction, subcellular trafficking and overall expression. Emerging evidence demonstrates that protein S-nitrosylation, the covalent attachment of a nitric oxide moiety to specified cysteine thiol groups, of GPCRs and/or their associated effectors also participates in the fine-tuning of receptor signaling and expression. This newly appreciated mode of GPCR system modification adds another set of controls to more precisely regulate the many cellular functions elicited by this large group of receptors. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.     

Fluorophore-tagged GPCR ligands

Fluorescently tagged drug molecules can be successfully employed to visualize the location of their receptor target at the single-cell level. Furthermore, if their binding to the receptor is reversible, one can now obtain detailed pharmacological information such as affinity using single-molecule detection techniques. When coupled to the growing exploitation of fluorescence-based read-outs in high throughput and high content screening, it is clear that fluorescent molecules offer a safer, more powerful and more versatile alternative to radioligands in molecular pharmacology and drug discovery. GPCR pharmacology has benefited enormously from the application of fluorescence-based technologies and we now possess a much greater understanding of this receptor family's basic molecular mechanisms of action through the careful design and judicious use of fluorescent peptide and small-molecule-based ligands.     

Proton transfer-mediated GPCR activation

G-protein coupled receptors (GPCRs) play essential roles in signal transduction from the environment into the cell. While many structural features have been elucidated in great detail, a common functional mechanism on how the ligand-binding signal is converted into a conformational change on the cytoplasmic face resulting in subsequent activation of downstream effectors remain to be established. Based on available structural and functional data of the activation process in class-A GPCRs, we propose here that a change in protonation status, together with proton transfer via conserved structural elements located in the transmembrane region, are the key elements essential for signal transduction across the membrane.     

Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment

The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with?a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast,?CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.     

Pharmacogenomics of GPCR Drug Targets

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A hypothesis for GPCR activation

Growing evidence that rhodopsin (RD) and related G protein-coupled receptors form functional dimers/oligomers, followed by direct proof (using atomic force microscopy) that in the retina disc membrane RD associates into a paracrystalline network of rows of dimers, need models of the RD-transducin (Gt) complex that would envision an optimal RD dimer/oligomer able to satisfy all well-documented interactions with Gt. Of the models proposed so far, only a few refer to RD dimers and only one of them proposes a complex of Gt with an RD oligomer (Filipek S, Krzyko KA, Fotiadis D, Liang Y, Saperstein DA, Engel, A, Palczewski K Photochem Photobiol Sci 3: 628–638, 2004). This paper puts forward a hypothesis on another arrangement of RD monomers into the reported network of rows of dimers. Arguments for the compatibility of this set-up with interactions and activation of RD in the complex with Gt, in particular, with the well-documented movement of transmembrane helix 6 and cytosolic loop 3, which is vital for RD activation, are provided and discussed.This revised version was published online in June 2005 with corrections to the acknowledgements.     

Ubiquitously expressed GPCR membrane-trafficking orthologs

Olfactory receptors are a diverse set of G-protein-coupled receptors (GPCRs) that localize to cellular plasma membranes in the olfactory epithelium. Associated trafficking proteins often assist in targeting these GPCRs to the membrane, facilitating function. One such trafficking protein has been isolated as a mutant defective for both odorant response and proper receptor localization in Caenorhabditis elegans. This gene (ODR-4) allows for functional expression of olfactory receptors in heterologous cells that are otherwise incapable of targeting. We have isolated a full-length human cDNA that is homologous to the C. elegans gene at the protein level across nearly the entire gene by using a novel RecA-based gene enrichment procedure. This sequence is homologous to a family of orthologs that share predicted structural features, indicating a conserved function. The gene was expressed in 41 of 44 human, mouse, and rat tissues, suggesting an important role in trafficking olfactory and other GPCRs.     

Advances in GPCR Modeling Evaluated by the GPCR Dock 2013 Assessment: Meeting New Challenges

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GPCR signalling to the translation machinery

G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and β-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a “GPCR signature” impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.     

Regulation of GPCR signaling in Hypertension

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65 million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.     

DEP-domain-mediated regulation of GPCR signaling responses

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response.