G protein amino-terminal alpha i2/alpha s chimeras reveal amino acids important in regulating alpha s activity

Gs and Gi2 are heterotrimeric G proteins that stimulate and inhibit, respectively, the activity of a common effector, adenylyl cyclase. The Gs and Gi2 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 alpha 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 alpha 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.