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

Abstract

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 ␤2 adrenergic receptor (␤2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The ␤2AR 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 ␤2AR 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 ␤2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the ␤ 2 AR and the structural basis for ligand efficacy.adrenergic ͉ constitutive activity ͉ receptor ͉ conformation ͉ inverse agonist

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

“…Indeed, the G protein in its empty form is well known to stabilize the high agonist affinity state of GPCRs and then the active state of the receptor (44). Consistent with this model, we found that overexpressing the Gαo dominant negative mutant that displays a low affinity for both GDP and GTP has a direct effect on the ECD dimer dynamics and, indeed, increases agonist potency.…”

β-arrestin–biased signaling through the β 2 -adrenergic receptor promotes cardiomyocyte contraction

“…TM6 movement associated with receptor activation and G-protein interaction has been previously monitored using purified β 2 AR modified with monobromobimane at Cys 265 (mbb-β 2 AR) (28,29). The environmentally sensitive monobromobimane demonstrates a decrease in peak fluorescence and a red shift upon TM6 movement when Cys 265 moves from a local hydrophobic environment to a position that is solvent exposed (28).…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…For example, direct and indirect measures of the RG complex suggest that coupling can be increased by agonists (65-67) and decreased or prevented by antagonists (66,68). Also, the supposed distribution of receptors between uncoupled and coupled states appears to be shifted by mutations and environmental factors that favor the latter and increase constitutive activity (45, 65, 67, 69 -72).…”