PT - JOURNAL ARTICLE AU - X M Guan AU - A Amend AU - C D Strader TI - Determination of structural domains for G protein coupling and ligand binding in beta 3-adrenergic receptor. DP - 1995 Sep 01 TA - Molecular Pharmacology PG - 492--498 VI - 48 IP - 3 4099 - http://molpharm.aspetjournals.org/content/48/3/492.short 4100 - http://molpharm.aspetjournals.org/content/48/3/492.full SO - Mol Pharmacol1995 Sep 01; 48 AB - The beta 3-adrenergic receptor (beta 3AR) is a member of the super-family of G protein-coupled receptors that are characterized by seven putative transmembrane helices connected by hydrophilic loops. The mechanism by which the activated beta ARs transmit the signals across the plasma membrane involves the stimulation of Gs, which in turn activates adenylyl cyclase, yielding the second messenger cAMP. In the present study, we created a series of mutant beta 3ARs to explore the structural basis for the subtype-specific binding of BRL 37344, a beta 3-selective agonist, and for the coupling of the receptor to Gs. To study the mechanism of subtype-specific binding of BRL 37344, chimeric beta 2/beta 3ARs were constructed and expressed in Raji cells. Binding studies suggest that the transmembrane segment 5 region of the beta 3AR contains critical determinants for observed high affinity for BRL 37344. Previous studies of beta 2ARs have demonstrated a role for the third intracellular loop in activating Gs. To investigate the role of this region in the beta 3AR, we constructed mutant beta 3ARs lacking a small segment of the amino- or carboxyl-terminal domain of the third 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 [125l]iodocyanopindolol, the agonist-stimulated cAMP production mediated by these mutant receptors is markedly attenuated or abolished. In addition, both mutant beta 3ARs exhibit an approximately 10-fold increase in affinity for agonist binding, whereas the affinity for antagonists is not affected. This increased agonist affinity is not altered by treatment with 100 microM 5' quanylyl-imidodiphosphate, suggesting that these mutant receptors are uncoupled from G proteins. The results of the present study demonstrate that these regions of the third intracellular loop of beta 3AR are critical for coupling to G proteins and suggest a role for these regions in maintaining the resting state of the unliganded receptor.