Generation and analysis of constitutively active and physically destabilized mutants of the human beta(1)-adrenoceptor

• Published in: Molecular pharmacology 2002-09, Vol.62 (3), p.747-755
• Main Authors: McLean, Alison J, Zeng, Fu-Yue, Behan, Dominic, Chalmers, Derek, Milligan, Graeme
• Format: Article
• Language: English
• Subjects: Adrenergic beta-Antagonists - pharmacology; Betaxolol - pharmacology; GTP-Binding Protein alpha Subunits, Gs - metabolism; Guanine Nucleotides - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Humans; Mutation; Receptors, Adrenergic, beta-1 - genetics; Receptors, Adrenergic, beta-1 - metabolism; Sulfur Radioisotopes; Transfection; Up-Regulation
• ISSN: 0026-895X
• DOI: 10.1124/mol.62.3.747

Constitutive activity of wild-type and mutant forms of human beta(1)- and beta(2)-adrenoceptors was measured by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays using fusion proteins between these receptors and G(s)alpha. Constitutive activity of the beta(1)-adrenoceptor is enhanced by mutation of Leu(322). The ability of ligands to suppress receptor instability and produce up-regulation is often associated with constitutively active mutants. Leu(322)Lysbeta(1)-adrenoceptor, but not wild type, was up-regulated by exposure to the beta(1)-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the beta(1)-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the beta(2)-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the beta(1)-adrenoceptor with the equivalent alpha(1b)-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both beta(1)- and beta(2)-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu(322)Lys mutation into the alpha(1b)-adrenoceptor substituted beta(1)-adrenoceptor to produce the CAMKbeta(1)-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [(35)S]GTPgammaS binding. Arg(156)Alabeta(1)-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [(35)S]GTPgammaS binding to all the fusion proteins was increased substantially by isoprenaline. Despite the ability of betaxolol to cause up-regulation of many mutants, only for the CAMbeta(2)-adrenoceptor-G(s)alpha and CAMKbeta(1)-adrenoceptor-G(s)alpha fusion proteins was the basal binding of [(35)S]GTPgammaS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant beta-adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.