Alpha I1 Subunits Research Articles

Involvement of G-protein alpha il subunits in activation of G-protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors.

This study investigated the type of G-protein alpha subunit(s) that human neuropeptide Y (NPY)1 receptors preferentially utilize when activating G-protein gated K+ currents. Two electrode voltage-clamp recordings were made from Xenopus oocytes that had been injected with cDNAs encoding either human NPY1 or D2(short) dopamine receptors, and GIRK1 a cloned rat brain K+ channel. These receptors were also co-injected with G-protein alpha i1, alpha i2, alpha i3 and alpha o1 subunits to determine which subunit(s) modulate the efficiency of signal transduction. In NPY1 receptor injected cells neuropeptide Y (100 nM) caused a 53 +/- 10 nA inward current (n = 14; EC50 = 3 nM) and this effect was blocked by pertussis toxin (500 ng ml-1 24 h). Activation of GIRK1 currents by neuropeptide Y was selectively potentiated by alpha i1 subunit cDNA whereas coupling dopamine of D2 receptors to this channel was not.

Palmitoylation of a G protein alpha i subunit requires membrane localization not myristoylation.

Palmitoylation is a dynamic, post-translational modification of the amino terminus of heterotrimeric G protein alpha subunits. Since myristoylation, beta gamma interactions, and membrane attachment also involve the amino terminus of the G protein alpha i1 subunit, we studied the relationships between palmitoylation and these events. Using COS cell transfection, the turnover of palmitate was slower on alpha i1 subunits co-expressed with beta and gamma subunits than on the alpha i1 subunit expressed alone. Mutation of cysteine 3 of alpha i1 prevented [2H]palmitate but not [3H]myristate incorporation and decreased the membrane localization of this protein. This nonpalmitoylated mutant could form a heterotrimer with co-expressed beta gamma subunits which restored its membrane localization. A nonmyristoylated alpha i1 mutant (glycine 2 to alanine) could incorporate [3H]palmitate when co-expressed with beta gamma subunits and localized to the membrane. The [3H]palmitate turnover of this nonmyristoylated mutant was more rapid than seen with the wild-type alpha i1 subunit. While myristoylation is not required for palmitoylation, both myristoylation and beta gamma association can slow the turnover of palmitate on alpha i1. These results suggest that palmitoylation maintains the membrane attachment of the free alpha subunit and changes in beta gamma association could modulate palmitoylation during signaling.

Isoprenylation of an inhibitory G protein alpha subunit occurs only upon mutagenesis of the carboxyl terminus.

We transfected COS cells with expression vectors for the wild-type G protein alpha i1 subunit (pWT) and for mutated alpha i1 subunits, including the nonmyristylated glycine 2 to alanine mutant (pGA) and mutants in which the carboxyl termini of pWT and pGA were changed from CGLF to CVLS (pCVLS and pGA-CVLS, respectively). Immunoblot analysis of transfected COS cells with an antibody to residues 159-168 of the alpha i1 protein indicated that all four proteins were expressed. Unlike the WT and GA proteins, both CVLS mutant proteins failed to react with an antibody specific for the carboxyl terminus and failed to undergo pertussis toxin-catalyzed ADP-ribosylation. Analysis of COS cell lysates after [3H]mevalonic acid labeling indicated that specific incorporation of radioactivity occurred only in the alpha i1 subunits with the CVLS mutation. Immuno-precipitation of COS cell fractions after labeling with [35S]methionine indicated that both WT and CVLS mutant proteins were localized predominantly in the particulate fraction, whereas GA and GA-CVLS mutant proteins were found primarily in the soluble fraction. These results directly demonstrate that the carboxyl-terminal sequence, CGLF, is incapable of leading to isoprenylation but that alteration of two residues (glycine to valine, phenylalanine to serine) is sufficient to promote isoprenylation.

Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.

We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits.