FATTY ACYLATION OF ALPHA(Z) - EFFECTS OF PALMITOYLATION AND MYRISTOYLATION ALPHA(Z) SIGNALING

As the first step in an investigation of roles played by fatty acylati on of G protein alpha chains in membrane targeting and signal transmis sion, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three alpha subunits, At site I , only HA-tagged alpha(q) and alpha(z) functioned normally, alpha(s), alpha(q), and alpha(z) subunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activit y, Using epitope tagged alpha(z), we investigated effects of mutations in sites for fatty acylation, Mutational substitution of Ala for Gly( 2) (G2A) prevented incorporation of myristate and decreased but did no t abolish incorporation of palmitate. Substitution of Ala for Cys(3) ( C3A) prevented incorporation of palmitate but had no effect on incorpo ration of myristate, Substitution of Ala for both Gly(2) and Cys(3) (G 2AC3A) prevented incorporation of both myristate and palmitate. All th ree mutations substantially disrupted association of a, with the parti culate fraction. G(z)-mediated inhibition of adenylyl cyclase, trigger ed by activation of the D2-dopamine receptor, was, respectively, aboli shed (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhi b ition of adenylyl cyclase by a, was unchanged (G2AC3A), strongly dimin ished (G2A), or strongly enhanced (C3A), A nonacylated, mutationally a ctivated alpha(z) mutant inhibited adenylyl cyclase, although less pot ently than normally acylated, mutationally activated alpha(z). From th ese findings we conclude: (a) fatty acylations of alpha(z) increase it s association with membranes; (b) myristoylation is not required for p almitoylation of alpha(z) or for its productive interactions with aden ylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by alpha(z).