Rabbit Anti-Human RGS16 (Phospho-Tyr168) polyclonal antibody

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-HT1A receptor-(Cys(351)IIe) 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-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.

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell-cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gai, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.