Functional importance of the amino terminus of G

John R. Hepler, Gloria H. Biddlecome, Christiane Kleuss, Laura A. Camp, Sandra L. Hofmann, Elliott M. Ross, Alfred G. Gilman

Research output: Contribution to journalArticle

95 Scopus citations

Abstract

G is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified G, with palmitoyl-thioesterase in vitro failed to alter interactions of G with phospholipase C-β1, the G protein βγ subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated G (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of G. However, truncated G 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 G 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 G. 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 G caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of G. 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.

Original languageEnglish (US)
Pages (from-to)496-504
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number1
DOIs
StatePublished - Jan 5 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Hepler, J. R., Biddlecome, G. H., Kleuss, C., Camp, L. A., Hofmann, S. L., Ross, E. M., & Gilman, A. G. (1996). Functional importance of the amino terminus of G. Journal of Biological Chemistry, 271(1), 496-504. https://doi.org/10.1074/jbc.271.1.496