A model in which receptor dimers activate one G protein predicts that the two orthosteric binding sites will not behave identically. In this arrangement, binding of the G protein heterotrimer is asymmetric (Ga contacts one monomer, and the GPy subunit the second) and thus, its allosteric effects on ligand interaction differ between the two monomers. Agonist binding to the first orthosteric site will stabilize G protein association with the dimer in a defined orientation and thereby influence ligand affinity at the second site. If, as expected, allosteric stabilization of an agonist-r eceptor complex occurs through Ga, this explains activation of only one receptor protomer per dimer and predicts negative cooperative binding of orthosteric ligands, leading also to a single bound ligand per dimer .54, 111, 124, 130] . Indeed, ligand binding assays can suggest this cooperativity, for example, through differences in receptor number measured by saturation binding of two ligands  and alterations in the shape of saturation and competition isotherms [114, 132]. For a limited number of GPCRs, the best evidence for negative cooperativity is the enhancement of radioligand off rates by excess unlabeled ligand [114, 130, 132-134]. Effects on dissociation kinetics cannot be accounted for by heterogeneous receptor populations, and a significant proportion of receptor dimers must be present in the membrane for their observation. Even here it is necessary to be cautious, particularly as most of the current studies use peptide or protein ligands where binding may be a multistep process— similar results could be obtained if ligands compete for independent parts of a single large orthosteric "pocket." Additional complications include the fact that some GPCR ligands in fact display positive cooperativity [129, 132, 135], and both types of cooperativity may be transient and dependent on the effector . For example, the presence of G protein eliminates the positive cooperativity between neurotensin binding sites in the NTS1 receptor dimer . However, wider application of kinetic experiments to a spectrum of GPCR ligands would help define the extent of cooperative interactions between orthosteric sites and, by implication, the prevalence of GPCR dimers .
Eventually, a conceptual framework involving GPCR dimers may also provide new insight into the pharmacology of both allosterism and dual efficacy. For example, the mapped binding sites on the class A ghrelin receptor for orthosteric (ghrelin) and allosteric ligands (L-692,429) overlap significantly, yet they must bind simultaneously to exert their actions [10, 137] . An attractive explanation, among others, is for each ligand to bind separate protomers in a ghrelin receptor dimer, analogous to class C allosteric interactions -54, 138] . This is particularly the case if negative cooperativity ensures the second protomer site does not bind ghrelin  . We also know little about the mode of binding of other effectors to a potential receptor dimer, such as the arrestins. A popular model, based on inactive (and therefore noninteract-ing) structures of rhodopsin and arrestin, suggests that the two arrestin binding domains associate with different GPCR monomers in 2:1 stoichiometry . However, the most detailed investigations on the binding of radiolabeled arrestins to different receptors  or the interactions between rhodopsin and arrestin in rods  provide convincing data on the association of one GPCR with one arrestin molecule. Here, the allosteric influence of arrestin on a GPCR dimer will be symmetric, and in marked contrast to the effects of G proteins, agonist binding to both ligand binding sites should be stabilized. Immediately, it becomes apparent that ligand binding to GPCR dimer stabilized by a G protein (with negative cooperativity between sites) is qualitatively different from a symmetric ternary complex containing arrestin, providing another mechanism for ligand-directed signaling to associated pathways. Resolution of such issues requires more work on the stoichiometry of GPCR interaction with different effectors, including a vital role for classical pharmacology techniques.
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