Trends in Pharmacological Sciences
OpinionThe apparent cooperativity of some GPCRs does not necessarily imply dimerization
Introduction
G-protein-coupled receptors (GPCRs) are classified into different subtypes 1, 2. The simplest are the rhodopsin-like class A receptors, which are characterized by a binding pocket for small ligands in the seven-helices transmembrane region. The most complex are the class C GPCRs, such as the metabotropic glutamate receptor, which are characterized by a large extracellular domain that associates in a constitutive homo- or heterodimer [3]. Class A GPCRs were originally considered to be monomeric, but subsequent functional biophysical, biochemical and pharmacological studies suggested that they could dimerize 4, 5. The relevance of these observations has, however, been questioned 6, 7, 8, 9. Moreover, the use of well-defined detergent micelles or nanodiscs has demonstrated that a single rhodopsin or a single β2-adrenoceptor molecule is capable of highly efficient coupling to a G protein 10, 11, 12.
Recently, Gurevich and Gurevich have thoroughly addressed the issue of GPCR dimerization from the structural point of view [13]. Here, we wish to complement this critical analysis by reassessing the evidence for dimerization that is based on pharmacological studies. In membrane preparations from cells expressing two closely related class A GPCRs, it is frequently observed that the binding of one ligand specific for the first receptor is affected by the addition of a second ligand acting on the second receptor. Depending on the nature of the ligands (i.e. agonist, inverse agonist), the apparent cooperativity can be negative or positive. Nevertheless, interference in ligand binding is usually considered to be a clue for the formation of GPCR heterodimers in which the two ligand-binding sites are coupled in an allosteric manner.
Before detailing several cases of apparent cooperativity, it is important to remember that, in membrane preparations, even the binding of a single agonist to a GPCR does not follow a simple bimolecular scheme. Pharmacologists are familiar with the GTP shift: a drop in the affinity of GPCRs for agonists induced by guanine nucleotides. This effect, which is still observed in preparation where GPCRs are unambiguously in a monomeric state (i.e. enwrapped in a nanodisc) [11], is the pharmacological indication that agonist-liganded GPCRs control the GDP/GTP cycle of G proteins (Figure 1). In part, the popular ‘ternary complex model’ accounts for the effect of G proteins on agonist affinity and is briefly summarized 14, 15. However, because this model does not detail the role of guanine nucleotides in the G-protein activation process and ignores the irreversible aspects of some reactions, we detail the catalytic model that has successfully accounted for the kinetics and stoichiometry of G-protein activation by class A GPCRs such as rhodopsin and the muscarinic receptor 16, 17. We argue that this model can explain the apparent ligand-binding cooperativity of class A GPCRs without implying any receptor dimer.
Section snippets
The ternary complex equilibrium model
The classical ‘ternary complex model’ [14] proposes that the receptor is in equilibrium between two states: R, the inactive conformation, and R*, the active conformation that activates the G protein. In the absence of ligand, R predominates. Agonists (A) bind only to R* and stabilize this active conformation, whereas inverse agonists bind only to R and stabilize the inactive conformation. It is a strict lock-and-key mechanism. Importantly, the model does not specify a mechanism for the
The ‘catalytic out-of-equilibrium’ kinetic model
A catalytic scheme was proposed in 1981 to account for the fast kinetics and high stoichiometry of transducin activation by rhodopsin 20, 21. This model was further developed with the identification of intermediate steps in the catalytic process and the observation that affinities were inadequate to describe the reaction flow 17, 22, 23. Later, a more elaborate form was proposed by Waelbroeck to describe the kinetics of muscarinic-receptor–G-protein coupling [16]. The catalytic model is
Apparent negative cooperativity might imply competition for the G protein
Class A GPCR pairs have often been assumed to form heterodimers on the basis of their apparent ligand-binding cooperativity. Two well-documented cases are that of the CCR2 and CCR5 chemokine receptors [28] and the μ- and δ-opioid receptors 29, 30, 31.
Let us consider two closely related class A GPCRs, say R1 and R2, that respond to specific agonists, A1 and A2, but are both active on the same type of G protein. The typical observation is that on membrane preparations in the absence of GTP the
How could the G-protein pool be limiting?
The relative stoechiometry between GPCRs and G proteins is difficult to address. Should we know the total number of G protein and receptors in a cell, we could not be certain that all potentially interact because subcompartmentalization should favor some interactions and exclude other ones. Obviously, the pool of G protein can be limiting when receptors have been overexpressed. Yet, even without overexpression, the G-protein pool seems usually to be smaller than that of their cognate receptors,
Apparent positive cooperativity of ligand binding to GPCRs
Some cases of positive cooperativity in ligand binding might also be explained by the catalytic model. If one receptor, say R1, has substantial constitutive activity, an inverse agonist acting on R1 can liberate the pool of G protein pre-coupled to R1, which becomes available to R2. This will increase the high-affinity-binding component of R2. Such an effect has been observed in the case of the μ- and δ-opioid receptors: the binding of an agonist of the μ-opioid receptor is of greater amplitude
Functional cooperativity in vivo: the case of an orphan receptor
Functional cooperativity of GPCR pairs in vivo should be difficult to explain by our model because the presence of GTP precludes the formation of nucleotide-free receptor–G-protein complexes. An interesting case, however, is that of GPR50, an orphan GPCR that has been suggested to inhibit the MT1 melatonin receptor through heterodimerization [34]. GPR50 and MT1 are closely related class A GPCRs and both are coupled to Gi. Co-expression of GPR50 with MT1 is shown to antagonize MT1 signaling in a
Conclusion
We have discussed three cases of apparent cooperativity between class A GPCRs that had been formerly presented as evidence for the existence of GPCR dimers. We argue that it is difficult to get a definitive proof of the existence of dimers from ligand-binding studies because an alternative explanation based on the formation of GPCR–G-protein complexes can be proposed. In this view, cooperativity is not due to an allosteric coupling of the two ligand-binding sites in a GPCR dimer, but rather to
Acknowledgements
We thank Cathy Jackson for comments on the manuscript.
References (39)
Metabotropic glutamate receptor 5 is a disulfide-linked dimer
J. Biol. Chem.
(1996)- et al.
GPCR monomers and oligomers: it takes all kinds
Trends Neurosci.
(2008) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins
J. Biol. Chem.
(2007)- et al.
How and why do GPCRs dimerize?
Trends Pharmacol. Sci.
(2008) A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor
J. Biol. Chem.
(1980)A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model
J. Biol. Chem.
(1993)Kinetic analysis of the activation of transducin by photoexcited rhodopsin. Influence of the lateral diffusion of transducin and competition of guanosine diphosphate and guanosine triphosphate for the nucleotide site
Biophys. J.
(1992)Oligomerization of μ- and δ-opioid receptors. Generation of novel functional properties
J. Biol. Chem.
(2000)Heterodimerization of μ- and δ-opioid receptors occurs at the cell surface only and requires receptor-G protein interactions
J. Biol. Chem.
(2005)Can different receptors interact directly with each other?
Trends Neurosci.
(1982)