Fatty Acylation of α2

As the first step in an investigation of roles played by fatty acylation of G protein α chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three α subunits. At site I, only HA-tagged αqand αzfunctioned normally. αs, αq, and αzsubunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged αz, we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly2(G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys3(C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Gly2and Cys3(G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of αzwith the particulate fraction. Gz-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by αzwas unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated αzmutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated αz. From these findings we conclude: (a) fatty acylations of αzincrease its association with membranes; (b) myristoylation is not required for palmitoylation of αzor for its productive interactions with adenylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by αz. As the first step in an investigation of roles played by fatty acylation of G protein α chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three α subunits. At site I, only HA-tagged αqand αzfunctioned normally. αs, αq, and αzsubunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged αz, we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly2(G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys3(C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Gly2and Cys3(G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of αzwith the particulate fraction. Gz-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by αzwas unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated αzmutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated αz. From these findings we conclude: (a) fatty acylations of αzincrease its association with membranes; (b) myristoylation is not required for palmitoylation of αzor for its productive interactions with adenylyl cyclase; (c) palmitoylation is not required for, but may instead inhibit, signaling by αz.