STRUCTURAL BASIS FOR RECEPTOR SUBTYPE-SPECIFIC REGULATION REVEALED BY A CHIMERIC-BETA(3)/BETA(2)-ADRENERGIC RECEPTOR

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (betaARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the betaAR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta2AR has a C terminus containing 11 serine and threonine residues representing potential sites for betaAR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta3AR, and they are in a nonfavorable context. The beta3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta3/beta2AR which comprised the beta3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta2AR. When cells expressing this chimeric beta3/beta2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta3AR, even when betaAR kinase was overexpressed. In contrast, the chimeric beta3/beta2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta3AR, the C-terminal tail of the beta2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.