Functional importance of the amino terminus of Gq alpha.
Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.
Hepler
J R
JR
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Biddlecome
G H
GH
Kleuss
C
C
Camp
L A
LA
Hofmann
S L
SL
Ross
E M
EM
Gilman
A G
AG
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D003902:Detergents; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010170:Palmitoyl-CoA Hydrolase / Q000378:metabolism; D054799:Phospholipase C beta; D018411:Spodoptera; D010738:Type C Phospholipases / Q000378:metabolism
D003902:Detergents; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010170:Palmitoyl-CoA Hydrolase; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins