title: Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human β1-adrenoceptor variants.
abstract: G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human β1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human β1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants.
authors.affiliation: From the Institute of Pharmacology and Toxicology, Technische Universität München, 80802 Munich, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ahles
authors.affiliation: From the Institute of Pharmacology and Toxicology, Technische Universität München, 80802 Munich, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Rodewald
authors.affiliation: the Rudolf Virchow Center for Experimental Biomedicine, Universität Würzburg, 97080 Würzburg, Germany.
authors.forename: Francesca
authors.initials: F
authors.lastname: Rochais
authors.affiliation: the Institute of Pharmacology and Clinical Pharmacy, Universität Marburg, 35043 Marburg, Germany, and.
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
authors.affiliation: From the Institute of Pharmacology and Toxicology, Technische Universität München, 80802 Munich, Germany, the German Center for Cardiovascular Research, Partner Site Munich Heart Alliance, 80802 Munich, Germany stefan.engelhardt@tum.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
mesh_terms: D001120:Arginine / Q000737:chemistry; D019390:Arrestins / Q000378:metabolism; D002318:Cardiovascular Diseases / Q000378:metabolism; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D011487:Protein Conformation; D034741:RNA, Small Interfering / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D034741:RNA, Small Interfering; D018342:Receptors, Adrenergic, beta-1; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D001120:Arginine; D000242:Cyclic AMP
keywords: Adrenergic Receptor; Cardiovascular Disease; FRET; G Protein-coupled Receptor (GPCR); Genetic Variation; SNP