G-PROTEIN AMINO-TERMINAL ALPHA(I2) ALPHA(S) CHIMERAS REVEAL AMINO-ACIDS IMPORTANT IN REGULATING ALPHA(S) ACTIVITY

G(s) and G(i2) are heterotrimeric G proteins that stimulate and inhibit, respectively, the activity of a common effector. adenylyl cyclase. The G(s) and G(i2) alpha subunit polypeptides, alpha(s) and alpha(i2), are 65% homologous in primary sequence. A series Of alpha(i2)/alpha(s) chimeras and alpha(s) point mutations were used to map sequences in the alpha(s) polypeptide that regulate alpha(s) activity. An amino-terminal region controlling the activation of alpha(s) was determined to reside within residues Lys-25 to Glu-101. Amino-terminal alpha(i2)/alpha(s) chimeras that disrupt this region in a(s) result in an activated alpha(s). In contrast, replacement of this entire alpha(s) sequence with the analogous alpha(i2) sequence produces a chimera whose activity is similar to that of the wild-type alpha(s) polypeptide. The regulation of alpha(s) activation by the amino-terminal sequence is independent of the intrinsic GTPase function. Inhibition of alpha(s) GTPase function by the mutation Gln-227 to leucine is additive with the amino-terminal chimera mutations. These mutations appear to independently alter the two rate-limiting steps in activation of the G protein a subunit, i.e., GTP hydrolysis and GDP dissociation allowing subsequent GTP binding. Within this region of alpha(s), Arg-42 is just amino-terminal to the G-1 sequence comprising part of the GDP/GTP binding pocket. The G- 1 sequence interacts with the alpha- and beta-phosphoryl groups of GDP and GTP. Mutation of alpha(s) Arg-42 to lysine has modest effects on alpha(s) activation, but when placed in the background of the glutamine to leucine mutation the alpha(sR42K+Q227L) Mutant polypeptide stimulates cAMP synthesis significantly more than observed with alpha(sQ227L) expression. Specific mutations in the amino terminus, therefore, have the ability to enhance alpha(s) activation by influencing the rate of adenylyl cyclase activation which is independent of GTPase activity.