Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q).
Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.
Evanko
D S
DS
Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Thiyagarajan
M M
MM
Wedegaertner
P B
PB
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D007295:Inositol Phosphates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D019308:Palmitic Acid; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs