TY - JOUR
T1 - Functional importance of the amino terminus of Gqα
AU - Hepler, John R.
AU - Biddlecome, Gloria H.
AU - Kleuss, Christiane
AU - Camp, Laura A.
AU - Hofmann, Sandra L.
AU - Ross, Elliott M.
AU - Gilman, Alfred G.
PY - 1996/1/5
Y1 - 1996/1/5
N2 - Gqα is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gqα, with palmitoyl-thioesterase in vitro failed to alter interactions of Gqα with phospholipase C-β1, the G protein βγ subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gqα (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gqα. However, truncated Gqα and the single Cys mutants activated phospholipase C-β1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gqα 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 Gqα. 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 Gqα caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gqα. 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.
AB - Gqα is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gqα, with palmitoyl-thioesterase in vitro failed to alter interactions of Gqα with phospholipase C-β1, the G protein βγ subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gqα (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gqα. However, truncated Gqα and the single Cys mutants activated phospholipase C-β1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gqα 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 Gqα. 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 Gqα caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gqα. 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.
UR - http://www.scopus.com/inward/record.url?scp=0030066476&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030066476&partnerID=8YFLogxK
U2 - 10.1074/jbc.271.1.496
DO - 10.1074/jbc.271.1.496
M3 - Article
C2 - 8550609
AN - SCOPUS:0030066476
SN - 0021-9258
VL - 271
SP - 496
EP - 504
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -