G protein-coupled receptor dimerisation: Molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

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.     

Cross-inhibition of angiotensin AT1 receptors supports the concept of receptor oligomerization

G protein-coupled receptors are cell surface receptors that mediate the effects of extracellular signals in the endocrine/paracrine and sensory systems. Experimental evidence is accumulating, which suggest that these receptors form dimers or higher order oligomers. The functional relevance of G protein-coupled receptor dimerization or oligomerization has been raised in a number of different processes, including ontogeny, internalization, ligand-induced regulation, pharmacological diversity and signal transduction of these receptors. Agonist-independent homo- and hetero-oligomerization of the angiotensin AT1 receptor has been reported, and it has been suggested that hetero-oligomerization with beta-adrenergic receptors leads to cross-inhibition of these receptors. Much less is known about the functional interactions between AT1 receptor homo-oligomers. The aim of the present study was to analyze the functional interactions between these homo-oligomers by determining the functions of normal, AT1 receptor blocker (candesartan) resistant (S109Y) and G protein coupling deficient (DRY/AAY) AT1 receptors (co-)expressed in COS-7 cells. Although we have found no evidence that stimulation of a G protein coupling deficient receptor could cross-activate co-expressed normal receptors, candesartan binding to a signaling deficient receptor caused cross-inhibition of co-expressed candesartan resistant AT1 receptors. Since the studied mutations were in the third intracellular helix of the receptor, the observed effects cannot be explained with domain swapping. These data suggest that AT1 receptor blockers cause cross-inhibition of homo-oligomerized AT1 receptors, and support the concept that receptor dimers/oligomers serve as the functional unit of G protein-coupled receptors.     

Methods to monitor the quaternary structure of G protein-coupled receptors

A wide range of approaches has been applied to examine the quaternary structure of G protein-coupled receptors, the basis of such protein-protein interactions and how such interactions might modulate the pharmacology and function of these receptors. These include co-immunoprecipitation, various adaptations of resonance energy transfer techniques, functional complementation studies and the analysis of ligand-binding data. Each of the available techniques has limitations that restrict interpretation of the data. However, taken together, they provide a coherent body of evidence indicating that many, if not all, G protein-coupled receptors exist and function as dimer/oligomers. Herein we assess the widely applied techniques and discuss the relative benefits and limitations of these approaches.     

Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using mu- and delta-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When mu- and delta-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing mu- and delta-receptors by the methods available. However, hetero-oligomers were identified by the ability to co-immunoprecipitate mu-receptors with delta-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed mu- and delta-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein. In this study, we provide, for the first time, evidence for the direct interaction of mu- and delta-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.     

G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions

G protein-coupled receptors are dynamically regulated. Such regulation is frequently associated with covalent posttranslational modifications, such as phosphorylation, and with regulatory elements. G protein-coupled receptor kinases and casein kinase 1alpha play key roles in agonist-dependent receptor phosphorylations. Cross-talk between different receptors frequently involves second messenger-activated proteins, such as protein kinase C and protein kinase A. There is some evidence indicating that such kinases may not only turn off receptors but also switch their coupling to different G proteins. Receptor tyrosine kinases may phosphorylate and regulate G protein-coupled receptors and recent evidence indicates that other kinases, such as Akt/protein kinase B and phosphoinositide 3-kinase, may participate in such regulations as integrators of signalling.Recent approaches have shed new light on G protein-coupled receptor interactions that provide novel mechanisms of action and regulation. G protein-coupled receptor activities go beyond G proteins and receptors can be partners of exquisitely assembled signalling complexes through molecular bridges composed of multidomain proteins. The possibilities of interaction increase enormously through the diversity of structural and functional domains present in complex proteins, many of them just known as predicted sequences.     

G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.     

Heterodimerization of type A and B cholecystokinin receptors enhance signaling and promote cell growth

Dimerization of several G protein-coupled receptors has recently been described, but little is known about its clinical and functional relevance. Cholecystokinin (CCK) and gastrin are structurally related gastrointestinal and neuronal peptides whose functions are mediated by two structurally related receptors in this superfamily, the type A and B CCK receptors. We previously demonstrated spontaneous homodimerization of type A CCK receptors and the dissociation of those complexes by agonist occupation (Cheng, Z. J., and Miller, L. J. (2001) J. Biol. Chem. 276, 48040-48047). Here, for the first time, we also demonstrate spontaneous homodimerization of type B CCK receptors, as well as heterodimerization of that receptor with the type A CCK receptor. Unlike type A CCK receptor dimers, the homodimerization of type B CCK receptors was not affected by ligand occupation. However, although heterodimers of type A and B CCK receptors bound natural agonists normally, they exhibited unusual functional and regulatory characteristics. Such complexes demonstrated enhanced agonist-stimulated cellular signaling and delayed agonist-induced receptor internalization. As a likely consequence, agonist-stimulated cell growth was markedly enhanced in cells simultaneously expressing both of these receptors. Our results provide the first evidence that heterodimerization of G protein-coupled receptors can form a more "powerful" signaling unit, which has potential clinical significance in promoting cell growth.     

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.     

Homodimerization and heterodimerization of S1P/EDG sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) binds to and signals through several members of a group of G protein-coupled receptors (GPCRs) known as the S1P/EDG family. Several of these receptors are coexpressed in various cell types and recent reports have shown that biological effects of S1P often require more than one S1P receptor subtype. Recent evidence indicates that many GPCRs exist as dimers. We show that S1P receptors form both homodimers as well as heterodimers with other members of the S1P subfamily of receptors. We also discuss the role that GPCR dimers play in receptor function and what this may mean for S1P signaling.     

Heterotrimeric G Proteins and the Single-Transmembrane Domain IGF-II/M6P Receptor: Functional Interaction and Relevance to Cell Signaling

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor’s structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.     

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.     

Protease signaling to G protein-coupled receptors: implications for inflammation and pain

Proteases, like thrombin, trypsin, cathepsins, or tryptase, can signal to cells by cleaving in a specific manner, a family of G protein-coupled receptors, the protease-activated receptors (PARs). Proteases cleave the extracellular N-terminal domain of PARs to reveal tethered ligand domains that bind to and activate the receptors. Recent evidence has supported the involvement of PARs in inflammation and pain. Activation of PAR(1), PAR(2), and PAR(4) either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Recent studies suggest a crucial role for the different PARs in innate immune response. The role of PARs in the activation of pain pathways appears to be dual. Subinflammatory doses of PAR(2) agonists induce hyperalgesia and allodynia, and PAR(2) activation has been implicated in the generation of inflammatory hyperalgesia. In contrast, subinflammatory doses of PAR(1) or PAR(4) increase nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as analgesic mediators. PARs have to be considered as an additional subclass of G protein-coupled receptors that are active participants to inflammation and pain responses and that could constitute potential novel therapeutic targets.     

Thematic Minireview Series: Cell Biology of G Protein Signaling

This thematic series is on the topic of cell signaling from a cell biology perspective, with a particular focus on G proteins. G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors) are typically found at the cell surface. Upon agonist binding, these receptors will activate a GTP-binding G protein at the cytoplasmic face of the plasma membrane. Additionally, there is growing evidence that G proteins can also be activated by non-receptor binding partners, and they can signal from non-plasma membrane compartments. The production of second messengers at multiple, spatially distinct locations represents a type of signal encoding that has been largely neglected. The first minireview in the series describes biosensors that are being used to monitor G protein signaling events in live cells. The second describes the implementation of antibody-based biosensors to dissect endosome signaling by G proteins and their receptors. The third describes the function of a non-receptor, cytoplasmic activator of G protein signaling, called GIV (Girdin). Collectively, the advances described in these articles provide a deeper understanding and emerging opportunities for new pharmacology.     

Metabotropic glutamate 1alpha and adenosine A1 receptors assemble into functionally interacting complexes

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.     

GIRK channel activation involves a local rearrangement of a preformed G protein channel complex

G protein-coupled signaling is one of the major mechanisms for controlling cellular excitability. One of the main targets for this control at postsynaptic membranes is the G protein-coupled potassium channels (GIRK/Kir3), which generate slow inhibitory postsynaptic potentials following the activation of Pertussis toxin-sensitive G protein-coupled receptors. Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), in intact cells, we provide evidence for the existence of a trimeric G protein-channel complex at rest. We show that activation of the channel via the receptor induces a local conformational switch of the G protein to induce channel opening. The presence of such a complex thus provides the means for a precise temporal and highly selective activation of the channel, which is required for fine tuning of neuronal excitability.     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.