Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment

Teresa L Z Jones, William F. Simonds, John J. Merendino, Mark R. Brann, Allen M. Spiegel

Research output: Contribution to journalArticle

161 Citations (Scopus)

Abstract

We transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, αs and αi1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled αs and αi were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected αi but could not be detected in αs even when it was overexpressed. We converted the second residue, glycine, of αi1 into alanine by site-directed mutagenesis. Upon transfection of the mutant αi1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal αi1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant αi1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant αi1 subunits. These results indicate that myristoylation is critical for membrane attachment of αi but not αs subunits.

Original languageEnglish (US)
Pages (from-to)568-572
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number2
StatePublished - 1990
Externally publishedYes

Fingerprint

COS Cells
Protein Subunits
Myristic Acid
GTP-Binding Proteins
Methionine
Membranes
Pertussis Toxin
Site-Directed Mutagenesis
Immunoprecipitation
Alanine
Glycine
Adenosine Diphosphate
Transfection
Complementary DNA
Peptides
Antibodies

Keywords

  • β-γ subunits
  • Immunoprecipitation
  • Signal transduction
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment. / Jones, Teresa L Z; Simonds, William F.; Merendino, John J.; Brann, Mark R.; Spiegel, Allen M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 87, No. 2, 1990, p. 568-572.

Research output: Contribution to journalArticle

@article{94c8c50f311c4cfd865bea0fdc8b6b8d,
title = "Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment",
abstract = "We transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, αs and αi1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled αs and αi were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected αi but could not be detected in αs even when it was overexpressed. We converted the second residue, glycine, of αi1 into alanine by site-directed mutagenesis. Upon transfection of the mutant αi1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal αi1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant αi1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant αi1 subunits. These results indicate that myristoylation is critical for membrane attachment of αi but not αs subunits.",
keywords = "β-γ subunits, Immunoprecipitation, Signal transduction, Site-directed mutagenesis",
author = "Jones, {Teresa L Z} and Simonds, {William F.} and Merendino, {John J.} and Brann, {Mark R.} and Spiegel, {Allen M.}",
year = "1990",
language = "English (US)",
volume = "87",
pages = "568--572",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "2",

}

TY - JOUR

T1 - Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment

AU - Jones, Teresa L Z

AU - Simonds, William F.

AU - Merendino, John J.

AU - Brann, Mark R.

AU - Spiegel, Allen M.

PY - 1990

Y1 - 1990

N2 - We transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, αs and αi1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled αs and αi were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected αi but could not be detected in αs even when it was overexpressed. We converted the second residue, glycine, of αi1 into alanine by site-directed mutagenesis. Upon transfection of the mutant αi1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal αi1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant αi1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant αi1 subunits. These results indicate that myristoylation is critical for membrane attachment of αi but not αs subunits.

AB - We transfected COS cells with cDNAs for the α subunits of stimulatory and inhibitory GTP-binding proteins, αs and αi1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled αs and αi were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected αi but could not be detected in αs even when it was overexpressed. We converted the second residue, glycine, of αi1 into alanine by site-directed mutagenesis. Upon transfection of the mutant αi1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal αi1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant αi1 could still interact with the β-γ complex, since purified βγ subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant αi1 subunits. These results indicate that myristoylation is critical for membrane attachment of αi but not αs subunits.

KW - β-γ subunits

KW - Immunoprecipitation

KW - Signal transduction

KW - Site-directed mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=0025101546&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025101546&partnerID=8YFLogxK

M3 - Article

VL - 87

SP - 568

EP - 572

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 2

ER -