Generation and Analysis of Constitutively Active and Physically Destabilized Mutants of the Human β1-Adrenoceptor
Open Access
- 1 September 2002
- journal article
- Published by Elsevier in Molecular Pharmacology
- Vol. 62 (3) , 747-755
- https://doi.org/10.1124/mol.62.3.747
Abstract
Constitutive activity of wild-type and mutant forms of human β1- and β2-adrenoceptors was measured by guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays using fusion proteins between these receptors and Gsα. Constitutive activity of the β1-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. Leu322Lysβ1-adrenoceptor, but not wild type, was up-regulated by exposure to the β1-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the β1-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the β2-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the β1-adrenoceptor with the equivalent α1b-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both β1- and β2-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu322Lys mutation into the α1b-adrenoceptor substituted β1-adrenoceptor to produce the CAMKβ1-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [35S]GTPγS binding. Arg156Alaβ1-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [35S]GTPγS 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 CAMβ2-adrenoceptor-Gsα and CAMKβ1-adrenoceptor-Gsα fusion proteins was the basal binding of [35S]GTPγS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant β-adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.Keywords
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