MUTATION OF VALINE RESIDUE UNIQUE TO ALPHA-SUBUNIT OF G(S) ABOLISHES ACTIVATION

We recently characterized a decapeptide sequence (residues 367-376) th at is important for the membrane association of the activated alpha su bunit of G(s). We report here that when this sequence is replaced by t he cognate sequence of G(i1) alpha a subunit, the chimeric protein (G( sis)alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of G(i1) alpha subunit, fully reproduces the loss of activation. Analysis of nucleotide interaction reveals that the mut ant V367T G(s) alpha protein poorly binds GDP or GTP. On the other han d, the conservative change of valine to isoleucine does not alter acti vation. Interestingly, members of the G(s) and G(12) classes have a va line and an isoleucine, respectively, at this position, whereas member s of the G(i) or G(q) class contain a threonine residue. The evolution ary relationship between the different classes suggests that the prese nce of a hydrophobic or a hydrophilic residue is not fortuitous in the se alpha subunits and might provide distinctive structural and/or func tional properties.