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

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subu nits. This GAP activity is mediated by the interaction of conserved residue s 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 activi ties of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increa sed GTP hydrolysis of G alpha(i1) by a similar extent, but with an about 10 0-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 glu tamate for compensation. G alpha(i1)K210E was insensitive to RGS16 but inte racted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(1)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89 K was without effect. Both G alpha(i1) and G alpha(i1) K210E mimicked the e ffect of alpha(2)-adrenoreceptor stimulation. G alpha(i1)K210E was sensitiv e 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 studie d 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 inh ibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude t hat 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 RG S proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing G al pha-RGS specificities in living cells.