G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives

Abstract

Most evidence indicates that, as for family C G protein-coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand-GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds.

Fig. 1.

Analysis of radioligand-binding experiments considering GPCRs as dimers: the two-state dimer model. For saturation experiments, K_D1 and K_D2 are the macroscopic equilibrium dissociation constants, which define the dissociation equilibria involved in the binding of a ligand to the receptor dimer. D_C represents the dimer cooperativity index. D_C = 0 implies no cooperativity, whereas positive and negatives values imply positive and negative cooperativity, respectively. For competition experiments, K_DB1 and K_DB2 correspond to the macroscopic equilibrium dissociation constants for the binding of the competing ligand to the first and second receptor in the dimer. K_DAB is a value of the association and dissociation of the competing ligand on a dimer semioccupied by the radioligand. Reciprocally, K_DBA is a macroscopic equilibrium dissociation constant of the radioligand binding to a receptor dimer semioccupied by the competing ligand. D_AB and D_BA represent the corresponding dimer radioligand/competitor modulation indexes. D_AB or D_BA = 0 implies no modulation, whereas positive and negatives values imply positive and negative modulation, respectively. D_CB defines a dimer cooperativity index for the competing ligand. D_CB = 0 implies no cooperativity, whereas positive and negative values imply positive and negative cooperativity.

Fig. 2.

Application of the two-state dimer model. Two different competing ligands, the adenosine A_2A receptor agonist CGS 21680 (A) and the A_2A receptor antagonist SCH-442416 (B and C), are used to displace the A_2A receptor antagonist [³H]ZM-241385 from membrane preparations of sheep striatum (A) or mammalian cells stably transfected with adenosine A_2A and A₁ receptors (B) or A_2A and dopamine D₂ receptors (C). (A) CGS 21680 displaces the binding of [³H]ZM-241385 in a biphasic manner; although not obvious by just looking at the graph, the analysis with a monomeric model gives a statistically significant better fit for two than for one binding site; analysis with the two-state dimer model indicates that the agonist does not show negative cooperativity (D_CB = 0); in fact, previous studies with saturation experiments with [³H]CGS 21680 usually show noncurvilinear Scatchard plots (Jarvis et al., 1989; Borea et al., 1995); the analysis nevertheless indicates that the biphasic displacement can be explained by an allosteric modulation between ligands, by which the binding of [³H]ZM-241385 facilitates the binding of CGS 21680 to the A2A receptor dimer (D_AB = 0.5). (B) Typical antagonist/antagonist competition, with SCH-442416 displacing in a monophasic manner [³H]ZM-241385 (D_CB = 0; D_AB = 0). On the other hand, in C, SCH-442416 displaces [³H]ZM-241385 in an obvious biphasic manner; the analysis with the two-state dimer model indicates that in this case, with coexpression of D₂ receptors, SCH-442416 binding to A_2A receptors displays a strong negative cooperativity (D_CB = −2.30; D_AB = 0). Results are modified from Casadó et al. (2009a) and Orru et al. (2011), where details of the methods, including radioligand concentrations, can be found. CGS 21680, 4-[2-[[6-amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid.