The regulator of G protein signaling RGS4 selectively enhances α2A-adreoreceptor stimulation of the GTPase activity of Go1α and Gi2α

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing G alpha(i1) and G alpha(i3) but markedly enhanced agonist-stimulation of the proteins containing G alpha(i2) and G alpha(o1). The effect of RGS4 on the alpha(2A)-adrenoreceptor-G alpha(o1) fusion protein was concentration-dependent with EC50 of 30 +/- 3 nM and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nM RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-G alpha(o1) or alpha(2A)-adrenoreceptor-G alpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-G alpha(o1) and alpha(2A)-adrenoreceptor-G alpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V-max and significantly increased K-m of the fusion proteins for GTP. The increase in K-m for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nM RGS4 was substantially higher for alpha(2A)-adrenoreceptor-G alpha(o1) than for alpha(2A)-adrenoreceptor-G alpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.