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Identification of key residues in transmembrane 4 responsible for the secondary, low-affinity conformation of the human 1-adrenoceptor

Baker, Jillian G.; Proudman, Richard G.W.; Hill, Stephen J.

Authors

JILLIAN BAKER jillian.baker@nottingham.ac.uk
Professor of Drug Discovery and Respiratory Medicine

Richard G.W. Proudman

Stephen J. Hill



Abstract

The ?1-adrenoceptor exists in two agonist conformations/states: 1) a high-affinity state where responses to catecholamines and other agonists (e.g., cimaterol) are potently inhibited by ?1-adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(?)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by ?1-adrenoceptor antagonists. Although both states have been demonstrated in many species (including human), the precise nature of the secondary state is unknown and does not occur in the closely related ?2-adrenoceptor. Here, using site-directed mutagenesis and functional measurements of production of a cyclic AMP response element upstream of a secreted placental alkaline phosphatase reporter gene and accumulation of 3H-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human ?1- and ?2-adrenoceptors, followed by single point mutations, to determine the key residues involved in the ?1-adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary ?1-adrenoceptor conformation. Thus, unlike at the human ?1-wild-type adrenoceptor, at ?1-TM4 mutant receptors, cimaterol and CGP12177 responses were both potently inhibited by antagonists. CGP12177 acted as a simple partial agonist with similar KB and EC50 values in the ?1-TM4 but not ?1-wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human ?1-wild-type adrenoceptors to a monophasic concentration response in the ?1-TM4 mutant receptors. Mutation of these amino acids to those found in the ?2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human ?1-adrenoceptor thus abolished this secondary conformation and created a ?1-adrenoceptor with only one high-affinity agonist conformation.

Citation

Baker, J. G., Proudman, R. G., & Hill, S. J. (2014). Identification of key residues in transmembrane 4 responsible for the secondary, low-affinity conformation of the human 1-adrenoceptor. Molecular Pharmacology, 85(5), https://doi.org/10.1124/mol.114.091587

Journal Article Type Article
Acceptance Date Mar 7, 2014
Publication Date May 31, 2014
Deposit Date Feb 13, 2017
Publicly Available Date Mar 28, 2024
Journal Molecular Pharmacology
Print ISSN 0026-895X
Electronic ISSN 1521-0111
Publisher American Society for Pharmacology and Experimental Therapeutics
Peer Reviewed Peer Reviewed
Volume 85
Issue 5
DOI https://doi.org/10.1124/mol.114.091587
Public URL https://nottingham-repository.worktribe.com/output/727980
Publisher URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132118/