Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of β2-adrenoceptor.
Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.
From the Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China, the Institute of Molecular Medicine, Centers for Life Sciences, Peking University, Beijing 100871, China, the Laboratory of Cardiovascular Science and yiuhowoo@pkusz.edu.cn.
Anthony Yiu-Ho
AY
Woo
the Department of Chemistry, Medical University of Lublin, Lublin, Poland.
Krzysztof
K
Jozwiak
the Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, and.
Lawrence
L
Toll
SRI International, Menlo Park, California 94025.
Mary J
MJ
Tanga
SRI International, Menlo Park, California 94025.
Joseph A
JA
Kozocas
SRI International, Menlo Park, California 94025.
Lucita
L
Jimenez
From the Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China, the Institute of Molecular Medicine, Centers for Life Sciences, Peking University, Beijing 100871, China.
Ying
Y
Huang
From the Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China, the Institute of Molecular Medicine, Centers for Life Sciences, Peking University, Beijing 100871, China.
Ying
Y
Song
the Department of Chemistry, Medical University of Lublin, Lublin, Poland.
Anita
A
Plazinska
the Department of Chemistry, Medical University of Lublin, Lublin, Poland.
Karolina
K
Pajak
Laboratory of Clinical Investigation, NIA, National Institutes of Health, Baltimore, Maryland 21224.
Rajib K
RK
Paul
Laboratory of Clinical Investigation, NIA, National Institutes of Health, Baltimore, Maryland 21224.
Michel
M
Bernier
Laboratory of Clinical Investigation, NIA, National Institutes of Health, Baltimore, Maryland 21224.
Irving W
IW
Wainer
the Institute of Molecular Medicine, Centers for Life Sciences, Peking University, Beijing 100871, China, xiaor@pku.edu.cn.
Rui-Ping
RP
Xiao
D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D055550:Protein Stability / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
C553443:ADRB2 protein, human; D058666:Adrenergic beta-2 Receptor Agonists; D018343:Receptors, Adrenergic, beta-2; D014443:Tyrosine; D048049:Extracellular Signal-Regulated MAP Kinases
Adrenergic Receptor; Cardiomyocyte Contraction; Cardiovascular; Functional Selectivity; G Protein-coupled Receptor (GPCR); Molecular Docking; Molecular Pharmacology; Signal Transduction; Site-directed Mutagenesis