title: Extracellular surface residues of the α1B-adrenoceptor critical for G protein-coupled receptor function.
abstract: Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs.
authors.affiliation: Institute for Molecular Bioscience (L.R., Å.A., R.J.L.) and School of Biomedical Sciences (W.G.T.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Lotten
authors.initials: L
authors.lastname: Ragnarsson
authors.affiliation: Institute for Molecular Bioscience (L.R., Å.A., R.J.L.) and School of Biomedical Sciences (W.G.T.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Åsa
authors.initials: Å
authors.lastname: Andersson
authors.affiliation: Institute for Molecular Bioscience (L.R., Å.A., R.J.L.) and School of Biomedical Sciences (W.G.T.), The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Walter G
authors.initials: WG
authors.lastname: Thomas
authors.affiliation: Institute for Molecular Bioscience (L.R., Å.A., R.J.L.) and School of Biomedical Sciences (W.G.T.), The University of Queensland, Brisbane, Queensland, Australia r.lewis@imb.uq.edu.au.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
mesh_terms: D058668:Adrenergic alpha-1 Receptor Antagonists / Q000494:pharmacology; D000409:Alanine / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D006224:Cricetinae; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D009638:Norepinephrine / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D011224:Prazosin / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D058668:Adrenergic alpha-1 Receptor Antagonists; D018340:Receptors, Adrenergic, alpha-1; D043562:Receptors, G-Protein-Coupled; D000409:Alanine; D009638:Norepinephrine; D011224:Prazosin