title: Enhanced detection of receptor constitutive activity in the presence of regulators of G protein signaling: applications to the detection and analysis of inverse agonists and low-efficacy partial agonists.
abstract: Fusion proteins between the human 5-hydroxytryptamine (5-HT)(1A) receptor and either wild type or certain pertussis toxin-resistant forms of G(o1)alpha and G(i1)alpha display constitutive GTPase activity that can be inhibited by the inverse agonist spiperone. Addition of recombinant regulator of G protein signaling (RGS) 1 or RGS16 to membranes expressing these fusion proteins resulted in elevation of this constitutive GTPase activity without significantly altering the binding affinity of antagonist/inverse agonist ligands. For a 5-HT(1A) receptor-(Cys(351)Ile)G(o1)alpha fusion protein the increase in basal GTPase activity was greater than 4-fold. Enzyme kinetic analysis demonstrated that the effect of RGS1 was as a GTPase-activating protein for the fusion construct. In the presence of the RGS proteins, both agonists and inverse agonists produced much more robust regulation of high-affinity GTPase activity than in their absence. This allowed detection of the partial agonist nature of WAY100635, which has been described previously as a neutral antagonist at the 5-HT(1A) receptor. Of a range of ligands studied, only haloperidol functioned as a neutral ligand in the presence of RGS1. These studies show that addition of a recombinant RGS protein provides a simple and novel means to elevate the fraction of basal membrane GTPase activity contributed by the constitutive activity of a receptor. By so doing, it also greatly enhances the ability to detect and analyze the effects of inverse agonists and to discriminate between neutral ligands and those with low levels of positive intrinsic efficacy.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Welsby
authors.forename: Elaine
authors.initials: E
authors.lastname: Kellett
authors.forename: Graeme
authors.initials: G
authors.lastname: Wilkinson
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D010879:Piperazines / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D020710:RGS Proteins / Q000378:metabolism / Q000502:physiology*; D011985:Receptors, Serotonin / Q000032:analysis*; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D020690:GTPase-Activating Proteins; D010879:Piperazines; D011506:Proteins; D011725:Pyridines; D020710:RGS Proteins; C107413:RGS16 protein; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins; C090413:N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; D019204:GTP-Binding Proteins