TY - JOUR

T1 - The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

AU - Yao, Xiao Jie

AU - Vélez Ruiz, Gisselle

AU - Whorton, Matthew R

AU - Rasmussen, Søren Gøgsig Faarup

AU - DeVree, Brian T

AU - Deupi, Xavier

AU - Sunahara, Roger K

AU - Kobilka, Brian

PY - 2009/6/9

Y1 - 2009/6/9

N2 - G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

AB - G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

KW - Adrenergic beta-2 Receptor Antagonists

KW - Bicyclo Compounds

KW - GTP-Binding Proteins

KW - Humans

KW - Ligands

KW - Protein Stability

KW - Receptors, Adrenergic, beta-2

KW - Signal Transduction

U2 - 10.1073/pnas.0811437106

DO - 10.1073/pnas.0811437106

M3 - Journal article

C2 - 19470481

VL - 106

SP - 9501

EP - 9506

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 23

ER -