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

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McLean AJ, Zeng FY, Behan D, Chalmers D, Milligan G

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

Mol Pharmacol. 2002 Sep;62(3):747-55.

PubMed ID

12181453 [ View in PubMed

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Abstract

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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Betaxolol	Beta-1 adrenergic receptor	Protein	Humans	Yes	Antagonist	Details