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 Citations (Scopus)

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
StatePublished - Jan 5 1996

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Palmitates
Lipoylation
Sf9 Cells
HEK293 Cells
Type C Phospholipases
Proteins
Palmitoyl-CoA Hydrolase
Protein Subunits
Cholinergic Receptors
Muscarinic Receptors
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
GTP-Binding Proteins
Detergents
Cytosol
Cholinergic Agents
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

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.

Functional importance of the amino terminus of G . / Hepler, John R.; Biddlecome, Gloria H.; Kleuss, Christiane; Camp, Laura A.; Hofmann, Sandra L.; Ross, Elliott M.; Gilman, Alfred G.

In: Journal of Biological Chemistry, Vol. 271, No. 1, 05.01.1996, p. 496-504.

Research output: Contribution to journalArticle

Hepler, JR, Biddlecome, GH, Kleuss, C, Camp, LA, Hofmann, SL, Ross, EM & Gilman, AG 1996, 'Functional importance of the amino terminus of G ', Journal of Biological Chemistry, vol. 271, no. 1, pp. 496-504.
Hepler JR, Biddlecome GH, Kleuss C, Camp LA, Hofmann SL, Ross EM et al. Functional importance of the amino terminus of G Journal of Biological Chemistry. 1996 Jan 5;271(1):496-504.
Hepler, John R. ; Biddlecome, Gloria H. ; Kleuss, Christiane ; Camp, Laura A. ; Hofmann, Sandra L. ; Ross, Elliott M. ; Gilman, Alfred G. / Functional importance of the amino terminus of G In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 1. pp. 496-504.
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abstract = "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.",
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AU - Ross, Elliott M.

AU - Gilman, Alfred G.

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