RT Journal Article
SR Electronic
T1 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
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1211
OP 1221
DO 10.1124/mol.61.5.1211
VO 61
IS 5
A1 Welsby, Philip J.
A1 Kellett, Elaine
A1 Wilkinson, Graeme
A1 Milligan, Graeme
YR 2002
UL http://molpharm.aspetjournals.org/content/61/5/1211.abstract
AB Fusion proteins between the human 5-hydroxytryptamine (5-HT)1A receptor and either wild type or certain pertussis toxin-resistant forms of Go1α and Gi1α 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-HT1Areceptor-(Cys351Ile)Go1α 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-HT1A 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.