Vol. 62, Issue 3, 747-755, September 2002

Generation and Analysis of Constitutively Active and Physically Destabilized Mutants of the Human ₁-Adrenoceptor

Alison J. McLean, Fu-Yue Zeng, Dominic Behan, Derek Chalmers, and Graeme Milligan

Molecular Pharmacology Group (A.J.M., G.M.), Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom; and Arena Pharmaceuticals (F.-Y.Z., D.B., D.C.), San Diego, California

Constitutive activity of wild-type and mutant forms of human ₁- and ₂-adrenoceptors was measured by guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding assays using fusion proteins between these receptors and G_s. Constitutive activity of the ₁-adrenoceptor is enhanced by mutation of Leu³²². The ability of ligands to suppress receptor instability and produce up-regulation is often associated with constitutively active mutants. Leu³²²Lys₁-adrenoceptor, but not wild type, was up-regulated by exposure to the ₁-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the ₁-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the ₂-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the ₁-adrenoceptor with the equivalent _1b-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both ₁- and ₂-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu³²²Lys mutation into the _1b-adrenoceptor substituted ₁-adrenoceptor to produce the CAMK₁-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [³⁵S]GTPS binding. Arg¹⁵⁶Ala₁-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [³⁵S]GTPS 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 CAM₂-adrenoceptor-G_s and CAMK₁-adrenoceptor-G_s fusion proteins was the basal binding of [³⁵S]GTPS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant -adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.