The regulator of G protein signaling RGS4 selectively enhances alpha(2A)-adreoreceptor stimulation of the GTPase activity of G(o1)alpha and G(i2)alpha

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G prot eins 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 agon ist stimulation of the fusion proteins containing G alpha(i1) and G alpha(i 3) but markedly enhanced agonist-stimulation of the proteins containing G a lpha(i2) and G alpha(o1). The effect of RGS4 on the alpha(2A)-adrenorecepto r-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 b y 100 nM RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to en hance 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 RG S4 amount and is consistent with previously proposed mechanisms of RGS func tion. Agonist-stimulated GTPase turnover number in the presence of 100 nM R GS4 was substantially higher for alpha(2A)-adrenoreceptor-G alpha(o1) than for alpha(2A)-adrenoreceptor-G alpha(i2). These studies demonstrate that al though RGS4 has been described as a generic stimulator of the GTPase activi ty of G(i)-family G proteins, selectivity of this interaction and quantitat ive variation in its function can be monitored in the presence of receptor activation of the G proteins.