G Protein–Coupled Receptor OligomerizationImplications for G Protein Activation and Cell Signaling

http://www.100md.com 《循环研究杂志》2004年第1期

     Abstract)9[+3g', 百拇医药

    The cardiovascular system is richly endowed with G protein–coupled receptors (GPCRs), members of the largest family of plasma membrane-localized receptors. During the last 10 years, it has become increasingly clear that many, if not all, GPCRs function in oligomeric complexes, as either homo- or hetero-oligomers. This review explores the mechanistic implications of GPCR dimerization and/or oligomerization on receptor activation and interactions with G proteins. The effects of GPCR oligomerization on receptor pharmacology, GPCR-mediated signaling, and potential contributions to GPCR crosstalk will be considered in the context of receptors important in the cardiovascular system. Our evolving understanding of the structural and functional consequences of GPCR oligomerization may provide novel and more selective sites for pharmacological tuning of cardiovascular function.)9[+3g', 百拇医药

    Key Words: G protein–coupled receptors oligomerization heterodimerization signal transduction G protein activation

    Introduction{m!, 百拇医药

    The largest family of plasma membrane–localized receptors is the superfamily of G protein–coupled receptors (GPCRs). GPCRs represent 1% of the human genome and are activated by an array of signals, from single photons to polypeptide hormones. Many receptors in the family are still "orphans," recognized in the genome by their characteristic serpentine, 7 transmembrane domain signature, but awaiting identification of their activating ligand. Reverse pharmacology screening strategies have permitted identification of an increasing number of receptor–ligand pairs. Interestingly, a significant fraction of newly identified ligands are peptides with cardiovascular effects, including apelin, orexins, ghrelin, urotensin, and relaxin.¹ Although characterization of orphan GPCRs will certainly contribute to our understanding of GPCR signaling in the cardiovascular system, the focus of this review is a newly recognized source of additional novel GPCR phenotypes, namely those resulting from GPCR oligomerization.

    There is now a large and diverse body of evidence (recounted in many recent reviews^2–7) that suggests that GPCRs do indeed function as dimers (or higher order oligomers), and that dimerization can occur among identical GPCRs, close family members, or GPCRs that are in distinct families. GPCR dimerization has documented effects on ligand binding, receptor activation, desensitization and trafficking, as well as receptor signaling. Mechanistic interpretation of these changes in GPCR function requires refinement of models for GPCR–G protein interactions. Although the genome provides a rich diversity of GPCRs, the potential for GPCR heterodimerization greatly increases the number of phenotypes that can be obtained from this already vast family of receptors, having implications for both cardiovascular physiology and the specificity of pharmacological interventions.z%9{?-7, http://www.100md.com

    Broad Evidence for GPCR Oligomerizationz%9{?-7, http://www.100md.com

    Experimental techniques have been crucial to establishing the prevalence and physiological significance of GPCR dimerization. Most present studies of GPCR oligomerization are performed using a combination of biochemical immunoprecipitation, functional studies, and/or resonance energy transfer techniques, performed in heterologous expression systems utilizing cloned and appropriately tagged (immunological epitopes or fluorescent reporters) GPCRs. A brief introduction to the most common methodologies and a survey of GPCRs found to be oligomerized by these methods are provided in the online data supplement available at http://www.circresaha.org; for more details, readers are referred to a number of comprehensive reviews.^2,8,9

    Contributions of GPCR Oligomerization to GPCR–G Protein Interactionsovq$*0p, 百拇医药

    Heterologous expression studies suggest that many, if not all, GPCRs form homo- and/or hetero-oligomers. Although these results will have to be verified on a case-by-case basis in vivo at physiological levels of GPCR expression, the current wealth of evidence supporting GPCR oligomerization requires a reconsideration of models for GPCR activation and GPCR/G protein coupling.ovq$*0p, 百拇医药

    The traditional view of GPCR/G protein coupling incorporates a monomeric GPCR interacting through specific intracellular domains (typically i2, i3, and/or proximal carboxyl terminal regions) with a single heterotrimeric G protein; agonist-induced conformational changes in the receptor, propagated through the transmembrane domain, ultimately result in exposure of critical residues at the GPCR/G protein interface that promote interaction with and activation of G proteins.^10,11 This "minimal model" is supported by countless mutagenesis studies of GPCRs in which the critical intracellular determinants for G protein activation have been empirically identified. Recent bioinformatics approaches have used this wealth of experimental data to attempt predictions of G protein coupling specificities from GPCR sequences.¹² From both experimental studies and bioinformatics, it is clear that the GPCR–G protein interaction surface is complex, and incorporates distinct regions of the GPCR, including the i2 loop, the N- and C-terminal portions of the i3 loop, and/or residues within the proximal portion of the carboxyl terminus (the so-called i4 loop anchored by lipid-modified cysteine residues). The potential for GPCR dimerization/oligomerization raises critical questions that impact on this minimal model for GPCR–G protein interactions, including (1) what is the nature of the GPCR dimer interface; (2) do allosteric interactions occur between partner GPCRs; (3) does agonist-mediated dimerization occur, and does it regulate the interaction of GPCRs with G proteins; and finally, (4) what is the stoichiometry of GPCR/G protein interactions?

    What Is the Nature of the GPCR Dimer Interface?*hz{k&, 百拇医药

    The nature of the dimer interface specifies not only which GPCRs can exhibit productive interactions, but influences models for potential allosteric interactions between dimer partners. Although many GPCRs have been shown to participate in homodimerization, heterodimerization is more variable, with some GPCRs exhibiting broad promiscuity,¹³ and others exhibiting a high degree of selectivity.¹⁴ Evidence to date suggests that there are distinctive dimerization interfaces or domains both within the transmembrane helices^15–18 as well as at either the extracellular amino terminus^15,19–21 or the intracellular carboxyl terminus,²² depending on the GPCR. Regardless of the domain responsible for initiation of dimerization, proximity of the transmembrane domains of the GPCR partners is assured. Family C GPCRs, CaR¹⁹ and metabotropic glutamate receptors mGluRs,²³ are stabilized by interdimer disulfide bonds localized at the extracellular, amino-terminal domain, whereas GABA_BRs interact via a coiled-coil domain at the carboxyl terminus.²⁴ m3 muscarinic receptors are also stabilized by disulfide bonds.²⁵ The majority of GPCRs, however, are stabilized as dimers/oligomers via noncovalent interactions. Two modes of interaction have been described (illustrated schematically in ), namely (1) contact dimerization, in which the relevant helix (helices) from one monomer contact(s) partner(s) in the other monomer, stabilizing the dimer pair; and (2) domain swapping, in which several helices from each receptor are "swapped" in the dimer, such that the functional monomer within the dimer contains helices contributed by both receptors (indicated by the distinct colors in ). Select examples of both types of interactions are available among GPCRs (see later), although the nature of the dimer interface for most GPCRs is unknown, and therefore the relative prevalence of the two modes remains to be established.

    fig.ommittedaa8f}/}, 百拇医药

    Figure 1. Potential GPCR dimer interfaces. Contact dimers, in which the interface between GPCR monomers involves surface contact between helices of two independent monomers (indicated by color), and domain-swapped dimers, in which helices 6 and 7 are exchanged or "swapped" between GPCR monomers (indicated by color), are illustrated, viewed at the cytoplasmic face of the membrane. Note the origins and locations of the intracellular loops (i1 loop, blue; i2 loop, black; i3 loop, red) contributing to the putative G protein binding surface in the two models.aa8f}/}, 百拇医药

    Contact site(s) have been identified for a very few GPCRs, and to date, only for homodimers. Experimental approaches include either (1) the use of synthetic peptides corresponding to various transmembrane helices to determine effects on receptor dimerization and/or activation, or (2) disulfide "trapping" in which cysteine residues are incorporated into suspected partner helices, and the propensity for disulfide bond formation is assessed. Synthetic peptides related to TM6 block both dimerization and activation of the ß₂AR,²⁶ whereas TM4 has been shown to mediate homodimerization of D₂ receptors by cysteine scanning mutagenesis and chemical crosslinking²⁷ and C5a receptors by disulfide trapping.¹⁸ TMs 1 and 2 were shown to be involved in yeast {alpha} -factor receptor dimerization by FRET analysis.¹⁵ A computational subtractive correlation method (based on the rhodopsin crystal structure, solvent accessibility, and location of residues on outer faces of helix bundles) has been applied to opioid receptor homodimers and heterodimers, and results (not yet experimentally verified) indicate a high degree of variability in interaction domains.^28,29 With respect to homodimers, TM4:TM4, TM5:TM5, or TM4:TM5 were predicted to be likely interfaces for OR, TM1:TM1 was most likely for µOR, whereas TM5:TM5 was most likely for {kappa} OR.²⁹ The model predicted that OR–µOR heterodimers were most likely stabilized by association of TM 4, 5, or 6 of the OR with TM1 of µOR, and accurately predicted no interaction(s) between µOR and {kappa} OR.²⁸ Evolutionary trace³⁰ and lipid-facing correlation models³¹ have also identified the most likely interfaces for representative GPCRs, but so far there has been little experimental data to verify the predictions on a GPCR family-wide scale. As additional GPCR pairings are established by experimental means, these models can be put to the test and refined. Because dimers as well as higher order oligomers have been characterized among GPCRs, it is highly likely that multiple forms of interactions will contribute to the stabilization of the final functional complex. In this regard, it is interesting to note the recent demonstration that mGluR1{alpha} receptors, which are constitutive disulfide-linked homodimers,²³ can be specifically coimmunoprecipitated with adenosine A₁ receptors from both HEK-293 cells and rat brain.³²

    The domain-swapping model was first suggested by the functional rescue upon coexpression of m3/{alpha} _2c and {alpha} _2c/m3 chimeric receptors, because binding of both m3 and _2c agonists was restored.³³ Although not established experimentally, domain swapping has also been invoked as an explanation for the altered ligand binding specificity of coexpressed and opioid receptors^28,34 because altered affinities (when compared with homodimers) can result when a functional binding unit comprises helices from two distinct receptor types (as illustrated by the color coding in ). It is theoretically possible to test domain swapping as the explanation for the altered agonist affinities of – opioid receptor heterodimers by generating chimeras between these two receptors containing helices 1 to 5 from one subtype and 6 to 7 from the other. Coexpression of these chimeras [(1–5)/(6–7) and (1–5)/(6–7)] should generate agonist affinities comparable to those seen with expressed – or – homodimers, because domain swapping should generate (1–5)/(6–7) plus {kappa} (1–5)/{kappa} (6–7) binding units.

    "Rescue" of receptor activity on coexpression of complementary inactivating mutants has also been suggested to result from domain swapping; in this case, one of the two "swapped" receptors contains the defective portions of both receptor mutants (and is nonfunctional), whereas the partner receptor is "reconstituted" with wild-type helices from each contributing receptor. For example, functional cooperation between partners in the ß₂AR homodimer has been inferred because nonpalmitylated, constitutively desensitized mutant ß₂AR (C341G–ß₂AR) can be rescued by coexpression with wild-type receptor.³⁵ The mutant and wild-type ß₂ARs assemble into functional heterodimers (as demonstrated by coimmunoprecipitation of epitope-tagged receptors), and have an activity and desensitization rate equivalent to wild-type homodimers.³⁵ Functional rescue was also observed when wild-type ß₂AR was coexpressed with a mutant lacking protein kinase A phosphorylation sites.³⁵

    Although there is clear evidence for domain swapping in specific cases (see review³⁰), systematic study of several other GPCRs has revealed no evidence for this mechanism.^36,37 Thus, it is likely that domain swapping may represent only one of several mechanisms utilized by particular GPCRs in dimer/oligomer formation and stabilization. It has been suggested that contact dimers and domain-swapped dimers have equivalent abilities to signal to G proteins, and the ability of a particular GPCR pair to form a functional dimer via either interaction depends on the relative energetics of the two possible pairings.³⁰n, 百拇医药

    Do Allosteric Interactions Occur Between Partner GPCRs?n, 百拇医药

    Binding of agonist to a GPCR causes conformational changes within the core of the helical transmembrane domain that are transmitted to the intracellular loops, resulting in G protein activation.^10,38 The presence of GPCRs in dimeric or oligomeric complexes makes allosteric interactions between the monomer partners within the dimer possible. The most straightforward example of allosteric interactions between dimer partners is the GABA_B receptor obligate heterodimer: agonist binding occurs only at the GABA_B1R amino terminal binding domain,³⁹ and G protein coupling has been mapped to the GABA_B2R intracellular domain.⁴⁰ Mutations in the GABA_B2R agonist binding domain do not affect receptor signaling³⁹ nor do mutations within the intracellular loops of the GABA_B1R^40–42inhibit G protein activation. This segregation of agonist binding on one monomer and critical G protein interactions on the other requires allosteric interactions between monomers at some level, either between agonist binding domains or between transmembrane helical domains. Interestingly, a chimera containing the functional domains of both receptors, ie, GABA_B1R extracellular domain plus GABA_B2R helical plus carboxyl terminal domains is not functional, whereas coexpression of the GABA_B1R/GABA_B2R plus GABA_B2R/GABA_B1R chimeras restores activity.⁴³ The requirement for allosteric coupling between subunits of the functional dimer is further supported by the ability of the GABA_B2R to increase the affinity of the GABA_B1R subunit for agonist, and to stabilize the closed (active) state of its agonist binding domain.^39,43 Likewise, although GABA_B1R does not productively interact with G proteins, the presence of the transmembrane plus intracellular domains of GABA_B1R is necessary for efficient GABA_B2R-mediated G protein activation.^40,43

    GABA_BR heterodimers also provide a provisional answer to a fundamental question regarding whether both receptors in a dimer must bind agonist for transduction, ie, G protein activation, to occur. With respect to GABA_BRs, only one receptor in the pair is competent to bind agonist³⁹ (see review⁴⁴) and therefore undergoes a conformational change to the closed state of the venus fly-trap module (extracellular agonist binding domain of family C members). Similar results have been derived from a crystal structure analysis of the dimerized extracellular venus fly trap module (ligand binding domain) of a related family C receptor, the metabotropic glutamate receptor, mGluR1.^21,23 Only one of the two binding modules "closes" on interaction with its agonist glutamate, triggering a global conformational change in both disulfide bond-linked partners,^21,23,44 and presumably causing propagated conformational changes to the respective transmembrane domains. Allosteric interactions can also be mediated by domains other than the transmembrane helices, as has recently been demonstrated for the membrane-proximal portion of the carboxyl terminus of calcium sensing receptors,^45,46 which can mediate cooperativity with respect to G protein activation.

    As described above, allosteric interactions have been identified for the major members of family C GPCRs, ie, GABA_BRs, mGluRs, and CaR. Does this reflect a general property of GPCRs in dimers/oligomers, or does this reflect a specific property of a highly restrictive group whose members function as obligate dimers? To answer this question, we need look no further than opioid receptors, members of family A (rhodopsin-like) GPCRs, which have been extensively characterized with respect to both homo- and heterodimerization. All three opioid receptors ({delta} , {kappa} , and µORs) have been shown to undergo homodimerization, and both – and –µ heterodimers have been demonstrated by coimmunoprecipitation^34,47 or BRET,⁴⁸ whereas {kappa} –µ heterodimers have not been observed.^34,49c\a.], 百拇医药

    Heterodimerization of opioid receptor subtypes has been evoked to explain the discrepancy between the number of known opioid receptors genes (, , and µ) and the pharmacologically distinct receptor subtypes characterized in vivo (₁, ₂, ₁, ₂, ₃, µ₁, and µ₂).^49,50 As discussed in the previous sections, both contact^28,29 and domain-swapping^28,34 models have been proposed to explain the changes in receptor function within the heterodimers, although neither model has been experimentally verified. Regardless of which mechanism is responsible for the interactions between monomers, the heterodimers formed by {delta} and {kappa} opioid receptors have pharmacological properties resembling the {kappa} ₂ receptor subtype characterized in vivo, ie, distinct from homodimers of either subunit. Not only does the {delta} –{kappa} heterodimer exhibit decreased affinities for either {delta} - or {kappa} -selective agonists and antagonists, but the heterodimer synergistically binds certain partially selective agonists with high affinity. Synergy with respect to agonist binding is also reflected in the activation of signaling pathways, both inhibition of adenylyl cyclase and phosphorylation of MAPK.³⁴ Similarly, µ–{delta} heterodimers exhibit reduced affinities for either µ- or {delta} -selective agonists, but synergistic interactions when both µ- and {delta} -selective agonists are applied simultaneously⁵¹ including enhanced affinities for endomorphin-1 and Leu-enkephalin,⁴⁷ suggesting alterations in the agonist binding site(s). Signaling via µ–{delta} heterodimers is insensitive to pertussis toxin, suggesting a shift in G protein preference from G_i to pertussis toxin–resistant subtypes.⁴⁷ Although all of these heterodimer effects on agonist binding and/or signaling can be interpreted as a result of domain swapping, they can also potentially reflect allosteric interactions between receptors within the homo- and heterodimers that are dependent on the location and/or specificity of the dimer interface.

    The occurrence of allosteric interactions between partner GPCRs may depend critically on the identity of the receptors. Here again, opioid receptors represent a well-studied case, because heterodimerization among opioid receptor subtypes evokes a variety of changes in agonist binding and/or G protein specificity that can be taken as a reflection of allosteric interactions. In sharp contrast, heterodimerization between opioid receptor subtypes and a variety of other family A GPCRs has also been noted, although none of the binding or signaling properties of the opioid receptor are altered, rather desensitization and/or internalization are affected. For example, both {delta} and µ opioid receptors are capable of forming heterodimers with ß₂AR. {delta} OR–ß₂AR heterodimers exhibit unaltered ligand binding properties and signaling, but activating either partner results in desensitization and internalization of the heterodimer complex.⁵² {kappa} OR homodimers do not undergo agonist-promoted endocytosis, and this phenotype is also dominant in {kappa} OR–ß₂AR heterodimers, which do not endocytose in response to either agonist, suggesting that heterodimerization in this case obscures domains critical to the process of ß₂AR internalization. Furthermore, ß₂AR-mediated phosphorylation of MAPK was reduced in cells expressing the {kappa} OR–ß₂AR heterodimer.⁵² µOR–sst_2A heterodimers displayed no alterations in ligand binding or signaling, but exhibited crossphosphorylation and crossdesensitization in response to agonists for either receptor.⁵³ Given the potential for distinct dimer interfaces between homo- and heterodimers of opioid receptors and their various partners, it is likely that some associations between GPCRs may represent a form of specific clustering or aggregation, which permits participants to be coimmunoprecipitated and cotrafficked (desensitized and/or internalized), but does not permit allosteric interactions between partners that could potentially alter agonist specificities or G protein signaling. A more intimate form of interaction between receptor partners, ie, what has been referred to as dimerization, may be required for allosteric interactions between receptor monomers. It should be stated that the dimer stoichiometry is a minimal one, and that higher order oligomeric complexes with distinct associations may occur, ie, (µOR:µOR):(sst_2a:sst_2a) tetramers in which distinct GPCR homodimers are associated in a cluster or aggregate would exhibit the same apparent stoichiometry via immunoprecipitation as the dimer µOR:sst_2a, although the functional consequences of the two types of association may be different. In this regard, it is interesting that recent studies have shown that GPCRs may associate with and traffic in oligomeric complexes with non-GPCR proteins. D1 dopamine receptors colocalize and interact (by BRET criteria) with NMDA NR1 subunits, and this interaction alters D1 dopamine receptor targeting to the plasma membrane as well as dopamine-induced sequestration.⁵⁴ Another study demonstrated colocalization and direct interactions between mGluR1 and voltage-sensitive calcium channels (Ca_v1.2) mediated by carboxyl terminal sites on the two proteins, resulting in enhanced regulation of calcium signaling in dendrites.⁵⁵ It seems likely that GPCRs will be found to undergo not only true dimerization (with attendant allosteric interactions), but also oligomerization, which alters targeting and/or provides the enhanced signaling and/or specificity normally thought to be promoted exclusively by scaffold proteins.

    Does Agonist-Mediated Dimerization/Oligomerization Occur, and Does It Regulate the Interaction of GPCRs With G Proteins?*y, 百拇医药

    Agonist binding to some GPCRs induces or stabilizes the dimerized state, which is the form of the receptor that productively interacts with G proteins. For other receptors, notably but not restricted to family C members, dimerization is constitutive and occurs in the endoplasmic reticulum. Examples of both types of associations are available, but the prevalence of each type of association within the GPCR superfamily is not yet known.*y, 百拇医药

    ßARs have been extensively characterized both biochemically and pharmacologically, and since the early 1980s, have been shown to purify as monomers or dimers (see review⁵⁶). ß₂AR dimerization has been studied by both coimmunoprecipitation of differentially epitope-tagged receptors²⁶ and by BRET.^57,58 By functional criteria, agonists have been shown to stabilize the dimeric state, whereas inverse agonists promote or stabilize the monomer.²⁶ The identity of transmembrane helix 6 as the dimerization interface and the importance of dimerization in ß₂AR signaling was underscored by inhibition of both dimerization and stimulation of adenylyl cyclase by addition of a peptide derived from transmembrane helix 6 (residues 276 to 296).²⁶ Thus, dimerization is required for efficient ß₂AR signaling, and can be promoted or inhibited by ligands. Agonist-induced oligomerization has also been reported for somatostatin receptors,^59,60 thyrotropin-releasing hormone receptors and gonadotropin-releasing hormone receptors,¹⁴ dopamine receptors,⁶¹ and heterodimers of adenosine A₁ and P2Y₁ receptors,⁶² whereas agonist-induced monomerization has been reported for cholecystokinin receptors⁶³ and {delta} OR.⁶⁴ Agonist-promoted changes in dimerization state may not be universal, and there are many examples of constitutive dimerization, including representative members of family C GPCRs (CaR, mGluRs, and GABA_BRs) (see review⁴⁴), vasopressin V1a and V2a,⁶⁵ oxytocin,⁶⁵ m3 muscarinic receptors,²⁵ melatonin receptors,⁶⁶ or neuropeptide Y receptors.⁶⁷ There are, however, divergent results for a number of GPCRs depending on the method used to determine dimerization. For example, by functional assay, ß₂ receptor dimerization has been shown to depend on agonist,²⁶ whereas via BRET, ß₁ or ß₂ homodimers and ß₁–ß₂ heterodimers have been shown to be constitutive.^57,58 Similar divergent results have been shown for {delta} OR (function and coimmunoprecipitation³⁴ versus BRET,^48,68 discussed in Levac et al⁴⁹), and CCR5 receptors (divergent results by coimmunoprecipitation^69,70 and BRET⁷¹). There are several likely sources of discrepancy between the two methods. First, the relative sensitivity of the two methods to the affinities between partner GPCRs, with coimmunoprecipitation requiring generally higher affinity interactions between partners to survive isolation procedures, whereas BRET requires affinities sufficient to stabilize proximity in vivo. Second, FRET/BRET depends on the distance between the two fluorescent tags within the complex, which may change as a result of ligand or regulator binding. Whether a difference in the strength of association between monomer partners is observed on agonist binding may depend on the magnitude of the conformational changes that the receptor monomers undergo, and the attendant changes in the distance between fluorescent tags. Differences in BRET resulting from agonist-induced conformational changes within constitutive complexes^14,66,72 have been described, whereas for other receptors, no change in BRET on agonist binding was observed.⁷¹ As more GPCRs are characterized with the explicit goal of understanding the contribution(s) of dimerization/oligomerization to their function, criteria for establishing constitutive versus regulated interactions among partners will of necessity be established.

    What Is the Stoichiometry of GPCR–G Protein Interactions?j, 百拇医药

    A critical question in GPCR signaling is the stoichiometry of GPCR–G protein interactions, although this question has been difficult to address because G protein–mediated signaling incorporates a catalytic component, ie, a single activated receptor molecule may serially activate a number of G proteins before agonist unbinding. Speculation that receptor dimers interacted with a single G protein heterotrimer began when the first crystal structure of a heterotrimeric G protein was solved: functional studies have mapped the many receptor interaction sites on G protein {alpha} and ß{gamma} subunits, and the data were not consistent with models incorporating the structure of a monomer of rhodopsin as the prototypical GPCR and the known structure of the G protein heterotrimer.^73–75 There are now several independent lines of evidence that suggest that G protein activation may require a dimeric GPCR interacting with a single heterotrimeric G protein.

    Recent studies on mammalian rhodopsins provides the most compelling physical evidence for an R₂:G({alpha} ß{gamma} ) stoichiometry. First, the x-ray crystal structure of bovine rhodopsin indicates that the cytoplasmic "footprint" of the transmembrane domain may be no more than 40 Å across,⁷⁶ which cannot account for interactions of the same monomer with both the transducin {alpha} subunit, and parts of the {gamma} subunit (for discussion, see Marshall⁷⁷). Second, a recent study utilizing atomic force microscopy found dimers to be the predominant form of mouse photoreceptor rhodopsin in isolated rod outer segment disk membranes, with the dimer interface at helices 4 and 5.⁷⁸ Finally, a number of functional studies have characterized cooperativity with respect to binding of transducin to rhodopsin, which could be explained by an R₂:G({alpha} ß{gamma} ) stoichiometry.^79,8071!#g, 百拇医药

    Strong biochemical evidence for an R₂:G({alpha} ß{gamma} ) stoichiometry comes from a recent study on leukotriene B₄ receptors.^81,82Optimization of methods for overexpression, recovery, and refolding of LTB₄R from bacterial inclusion bodies produced a soluble receptor that was appropriately refolded and bound both LTB₄ and structurally related antagonists with native affinities.⁸¹ The stoichiometry of LTB₄R:G(ß) protein interactions was determined by two independent methods; both chemical crosslinking in the presence of receptor, heterotrimeric G protein (G_i2ß₁₂), and agonist or solution phase neutron scattering indicated a pentameric stoichiometry, ie, R₂:G(ß).⁸² Conditions predicted to lead to low affinity of the receptor for G protein (GTP{gamma} S or antagonist) promoted the appearance of the monomeric species of receptor, leading to the minimal model⁸² (depicted generically in ), ie, agonist binding to a monomeric receptor promotes receptor dimerization, followed by interaction with heterotrimeric G protein (GDP-bound), ultimately resulting in GDP release, GTP binding, and dissociation of activated G protein subunits from the dimeric receptor.

    fig.ommitted&jtusy0, http://www.100md.com

    Figure 2. Minimal model for GPCR dimer–mediated G protein activation. Step 1 indicates potential binding modes for agonist to GPCRs, ie, either agonist binding to GPCR monomers drives dimerization or agonist binds to preexisting GPCR dimers (not shown). Loading of GPCRs with agonist induces conformational changes at the intracellular face of the receptor dimer, opening a pocket that can accommodate heterotrimeric G protein binding (step 2). G protein activation (step 3) results in dissociation of activated G protein subunits and recycling of the activated GPCR dimer (step 4a). Activated G protein interacts with effector(s) until GTP hydrolysis occurs on the subunit, which results in reassociation of the heterotrimer (step 4b).&jtusy0, http://www.100md.com

    A different type of evidence supporting GPCR dimers as the minimal functional unit comes from studies with membrane-tethered peptides, termed pepducins.^83,84 Peptides corresponding to the i3 loop of protease-activated receptors or melanocortin receptors that were membrane tethered (by either hydrophobic residues at the amino and carboxyl termini or by addition of a palmitate group) activated receptors in the absence of agonist.⁸⁴ Results from the studies were consistent with the tethered peptide and the monomeric receptor interacting to reconstitute a complete "dimeric" binding site for G proteins, presumably in the active conformation, permitting signaling in the absence of agonist.⁸³ Shortened peptides lacking the ability to induce receptor activation acted as antagonists, preventing agonist-mediated receptor activation.⁸⁴

    Finally, heterodimerization of a variety of GPCRs alters the G protein specificity of signaling (). Although a shift in G protein specificity can be explained by a number of mechanisms, examination of suggests that interaction of a single G protein with a receptor dimer can account for this data. That is, if the G protein interacts with the i2 or i3 loop(s), the composite heterodimer G protein binding site contains contributions from both GPCR partners, potentially altering the electrostatic binding surface and therefore G protein specificity.^11,8565otwm:, http://www.100md.com

    fig.ommitted65otwm:, http://www.100md.com

    Table 1. Functional Consequences of GPCR Heterodimerization65otwm:, http://www.100md.com

    Minimal Model for GPCR Dimer–Mediated Activation of G Proteins65otwm:, http://www.100md.com

    A minimal model consistent with the characteristics of GPCR dimerization discussed in the previous sections is depicted in . Agonist may bind to either monomeric receptor, promoting dimerization (step 1 in ), or the receptor dimer may be constitutive and agonist therefore binds to the appropriate site on the dimer (not shown). Further work with a wide variety of GPCRs in homo- or heterodimers is required to establish whether both agonist sites or only one must be occupied for productive interactions with G proteins; depicts all three possibilities (step 1), but for simplicity, further steps are depicted for only the doubly agonist occupied dimer. A single heterotrimeric GDP-bound G protein binds to a site composed of regions contributed by both GPCRs within the dimer (step 2), causing activation of the G protein (release of GDP and binding of GTP (step 3), generating free receptor and activated, dissociated G protein subunits. Dimeric receptor can recycle and activate other G proteins (step 4a), and G protein subunits ({alpha} -GTP and ß{gamma} ) can interact with various effectors until GTP hydrolysis occurs, resulting in reassociation of the heterotrimeric, inactive G protein (step 4b). Not depicted in the model are the potential allosteric interactions between receptors, which may alter agonist affinities on the two receptors and/or contribute to the conformational changes required to generate the active state needed for productive interactions with G proteins.

    Functional Consequences of GPCR Oligomerizationlo+kq, 百拇医药

    GPCRs have been extensively characterized both in vivo and in vitro with respect to receptor pharmacology, activation, and desensitization, as well as their role(s) in activation of cell signaling pathways. Although GPCR-mediated signaling is regulated at many cellular levels (see reviews^86,87), in this review, we consider the potential consequences of GPCR heterodimerization on GPCR function.lo+kq, 百拇医药

    Effects on GPCR Pharmacologylo+kq, 百拇医药

    Alterations in the agonist binding sites of GPCRs may accompany dimerization as a result of allosteric interactions or domain swapping between partners ). The general strategy of using a cell line stably expressing the GPCR of interest in screening for potent/specific pharmacophores can result in identification of compounds that are not as effective in vivo. Given the propensity of many GPCRs to heterodimerize, with attendant changes in binding site affinities, the promiscuity of some GPCRs may generate a heterogeneous receptor population that is cell-type specific. Iterative strategies in which likely GPCR partners in the tissue of interest are coexpressed with the target GPCR may be required to identify physiologically significant ligands. The ability of a partner to modify the binding affinity of a GPCR can potentially be used to clinical advantage, allowing development of agents specific for a given heterodimer pair to regulate a signaling pathway present in only a subset of tissues. For GPCRs that undergo agonist-mediated dimerization, stabilization of the dimer by bifunctional agonists could lead to greater specificity and efficacy of signaling.⁸⁸

    Effects on GPCR Signalingx22, 百拇医药

    GPCRs have been extensively characterized with respect to both ligand binding and activation of various signaling pathways. Widespread demonstrations of GPCR oligomerization raise interesting questions regarding which aspects of already well-characterized GPCR-mediated functions are potentially due to heterodimerization in vivo. Heterodimerization potentially alters G protein specificity, coupling to signaling pathways, and may also attenuate signaling (). It has been suggested that some GPCR heterodimers act as "coincidence detectors," synergistically increasing signaling when both agonists are present.³ Conversely, partners within heterodimers can negatively interact, attenuating signaling relative to the respective homodimers.⁸⁹ ß-Adrenergic receptors are a well-studied example relevant to the cardiovascular system. When expressed in HEK-293 cells, ß₁–ß₂AR heterodimers display hybrid signaling compared with the respective homodimers. ß₁–ß₂AR heterodimers stimulate adenylyl cyclase, to levels achieved by either ß₁ or ß₂AR homodimers.⁹⁰ In contrast, ß₂AR homodimers robustly stimulate ERK1/2 phosphorylation, whereas ß₁AR homodimers have no effect on this signaling pathway. ß₁–ß₂AR heterodimers are not able to activate the MAPK signaling cascade.⁹³ ß₂AR internalization is a prerequisite for activation of the MAPK cascade,⁹³ and heterodimerization with ß1AR limits ß₂AR interactions with arrestin and subsequent internalization. Cardiac myocytes express both ß₁- and ß₂AR, and selective up- and/or downregulation of receptor subtype expression accompanies a number of disease states. For example, during development of heart failure, ß₁AR expression is reduced whereas ß₂AR expression is increased, allowing selective increases in ERK1/2 signaling (discussed in Lavoie et al⁹⁰). In contrast to HEK-293 cells where coexpression of ß₁AR and ß₂AR results in attenuation of ERK1/2 signaling,⁹⁰ neonatal rat cardiomyocytes exhibit ß₁AR and ß₂AR signaling through adenylyl cyclase, as well as activation of ERK1/2,⁹¹ suggesting that in vivo segregation of receptors to distinct microdomains, eg, caveolae, may limit the extent of heterodimerization.⁹² Heterodimerization of ßARs has also been demonstrated with opioid receptor subtypes ({delta} and {kappa} )⁵² and {alpha} _2A receptors.⁹³ A particular GPCR may thus exhibit variable coupling to signaling pathways as a result of heterodimerization with diverse GPCR partners and/or as a result of subcellular segregation of receptors, which may also be specified by the partner GPCR.

    Contributions to GPCR Crosstalkv?u}i|i, 百拇医药

    It has become increasingly clear that GPCR signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather results from the complex interactions of multiple, branched signaling pathways, ie, signaling networks. Positive and negative feedback in such pathways, activated by multiple GPCRs, is termed crosstalk (see reviews^94–96). Unrecognized heterodimerization between distinct GPCRs can generate signaling phenotypes that may be interpreted as signaling pathway crosstalk. Crosstalk between signaling pathways regulating the cardiovascular system is often observed, and in general, is thought to result from the intersection of signaling pathways at multiple levels within signaling networks (see comprehensive review⁹⁷). It is highly likely that some well characterized examples of receptor crosstalk in the cardiovascular system will be the result of direct heterodimerization between the GPCRs involved, eg, the well-characterized crosstalk between {delta} OR and ß₁AR, which results in attenuation of myocardial responses to stress,^98,99 may very well be the result of receptor heterodimerization.

    A broad illustration of the potential contributions of GPCR heterodimerization in receptor crosstalk is available from recent studies on the angiotensin receptor. Two subtypes of angiotensin receptors have been identified, type 1 (AT₁) and type 2 (AT₂), each with distinct signaling properties.¹⁰⁰ Negative crosstalk has been observed when both AT₁ and AT₂ receptors are expressed in vascular smooth muscle cells, with AT₂ receptors inhibiting AT₁-mediated cell growth.^96,100 Most of the physiological effects of angiotensin II are mediated by AT₁ receptors (AT_1A and AT_1B). AT₁ receptors are noncovalent dimers¹⁰¹; allosteric interactions between the monomers within the homodimer has been demonstrated by coexpression studies with deficient mutants, which restores a normal binding site and signaling.¹⁰² Whereas G protein coupling and signal transduction via AT₁ receptors is well characterized, less is known about AT₂ receptors, although coexpression with AT₁ receptors often attenuates AT₁ responses.^96,100 Recent studies have demonstrated heterodimerization of AT₁ and AT₂ receptors both in vivo^103–105 and in vitro,^104,105with attendant inhibition of AT₁ receptor signaling, suggesting that AT₂ receptors act as a dominant-negative or antagonist of AT₁ receptor function,¹⁰⁴ ie, the negative crosstalk defined in vivo may result directly from heterodimerization of AT₁ and AT₂ receptors. Amino acids within the AT₂R third intracellular loop inhibit AT₁R-mediated IP₃ generation, and mutations of the AT₂R residues to their AT₁R counterparts are sufficient to permit AT₂ receptor–mediated generation of IP₃.¹⁰⁶ Both AT₁ and AT₂ receptors are expressed in many of the same tissues, and thus AT₁R signal attenuation may depend on relative AT₁/AT₂ receptor expression levels.

    AT₁ receptors also exhibit positive crosstalk in vivo as a result of heterodimerization, in this case with bradykinin B₂ receptors. Heterodimerization between AT₁ and B₂ receptors potentiates AT₁ receptor signaling and sequestration after activation.¹⁰⁷ Angiotensin II is a more potent activator of AT₁ receptors within AT₁:B₂ heterodimers, whereas the potency and efficacy of bradykinin is reduced. The enhancement of AT₁R signaling within the heterodimer does not require activation of the B₂ receptor, but does require the ability of the B₂ receptor to couple to G proteins.¹⁰⁷ AT₁ and B₂ receptors are coexpressed in smooth muscle,¹⁰⁸ kidney,¹⁰⁹ platelets, and omental vessels,¹⁰⁵ and thus AT₁:B₂ heterodimers may contribute to the normal functioning of the renin-angiotensin system, as suggested by alterations observed in preeclampsia. A consistent feature of preeclampsia, a multifactorial disorder characterized by hypertension and proteinuria during pregnancy,¹¹⁰ is an increased vascular responsiveness to angiotensin II, which is not the result of increased circulating levels of the hormone nor its receptor AT₁.¹¹¹ A partial explanation for the increased responsiveness to angiotensin II is the 4- to 5-fold increase in B₂ receptor expression in both platelets and omental vessels from women with preeclampsia, but not from normotensive pregnant women.¹⁰⁵ The increase in B₂ receptor expression in platelets and omental vessels leads to an increase in AT₁-B₂ receptor heterodimerization; platelets from preeclamptic women displayed enhanced signaling in response to angiotensin II, despite levels of AT₁ receptor comparable to normotensive women. Interestingly, AT₁-B₂ heterodimers were insensitive to reactive oxygen species, which inactivate AT₁ receptor homodimers. The increased responsiveness to angiotensin II in women with preeclampsia results from both increases in AT₁-B₂ heterodimer formation and from the resistance to inactivation by H₂O₂ of AT₁ receptors within heterodimers.¹⁰⁵ Preeclampsia is thus the first physiological disorder linked to altered levels of heterodimerization of G protein–coupled receptors, and a prime example of crosstalk at the level of the receptors themselves.

    Importance for Cardiovascular Physiology&4e1#, 百拇医药

    The last 10 years has seen a revolution in our understanding of GPCR structure and function, with a growing awareness of the contributions of homo- and heterodimerization to the generation of novel receptor phenotypes. GPCR heterodimerization can result in altered ligand selectivities and distinctive coupling to signal transduction pathways, providing an additional source of richness for the fine-tuning of cardiovascular physiology, as well as potentially novel targets for selective pharmacological interventions.&4e1#, 百拇医药

    Acknowledgments&4e1#, 百拇医药

    This work was supported by NIH GM 58578 and Novartis Pharma, AG. I thank Dr Catherine H. Berlot for helpful comments on the manuscript.&4e1#, 百拇医药

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