Polarity exchange at the interface of regulators of G protein signaling with G protein α-subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and G alpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with G alpha(t) and G alpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of G alpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in G alpha(i1), this lysine was changed to glutamate for compensation. G alpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(1)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both G alpha(i1) and G alpha(i1) K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. G alpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than G alpha(i1). Analogous mutants of G alpha(q) (G alpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type G alpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of G alpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in G alpha(i) and G alpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing G alpha-RGS specificities in living cells.