DETERMINATION OF STRUCTURAL DOMAINS FOR G-PROTEIN COUPLING AND LIGAND-BINDING IN BETA(3)-ADRENERGIC RECEPTOR

The beta(3)-adrenergic receptor (beta(3)AR) is a member of the superfa mily of G protein-coupled receptors that are characterized by seven pu tative transmembrane helices connected by hydrophilic loops. The mecha nism by which the activated beta ARs transmit the signals across the p lasma membrane involves the stimulation of G(s), which in turn activat es adenylyl cyclase, yielding the second messenger cAMP. In the presen t study, we created a series of mutant beta(3)ARs to explore the struc tural basis for the subtype-specific binding of BRL 37344, a beta(3)-s elective agonist, and for the coupling of the receptor to G(s). To stu dy the mechanism of subtype-specific binding of BRL 37344, chimeric be ta(2)/beta(3)ARs were constructed and expressed in Pail cells. Binding studies suggest that the transmembrane segment 5 region of the beta(3 )AR contains critical determinants for observed high affinity for BRL 37344. Previous studies of beta(2)ARs have demonstrated a role for the third intracellular loop in activating G(s). To investigate the role of this region in the beta(3)AR, we constructed mutant beta(3)ARs lack ing a small segment of the amino- or carboxyl-terminal domain of the t hird intracellular loop, Expression of these mutant receptors in mouse L cells and Raji cells reveals that although both mutants are capable of binding the antagonist [I-125]iodocyanopindolol, the agonist-stimu lated cAMP production mediated by these mutant receptors is markedly a ttenuated or abolished. In addition, both mutant beta(3)ARs exhibit an approximately 10-fold increase in affinity for agonist binding, where as the affinity for antagonists is not affected. This increased agonis t affinity is not altered by treatment with 100 mu M 5'quanylyl-imidod iphosphate, suggesting that these mutant receptors are uncoupled from G proteins. The results of the present study demonstrate that these re gions of the third intracellular loop of beta(3)AR are critical for co upling to G proteins and suggest a role for these regions in maintaini ng the resting state of the unliganded receptor.