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In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.

Publication ,  Journal Article
Higgins, JB; Casey, PJ
Published in: J Biol Chem
March 25, 1994

The gamma subunits of the heterotrimeric G proteins (G gamma) are subject to carboxyl-terminal processing. This processing involves prenylation of a cysteine residue initially 4 amino acids from the carboxyl terminus, endoproteolytic truncation of the 3 terminal amino acids, and methylation of the now carboxyl-terminal prenylcysteine residue. The significance of each of these modifications in the ultimate properties of G proteins is not yet clear. We have developed in vitro systems for the production of the three processing intermediates (unmodified, prenylated, and truncated-prenylated) for two G gamma subunits, one which is subject to farnesylation (G gamma 1) and one which is geranylgeranylated (G gamma 2). Assessment of the functional consequences of the processing of G gamma was found to require reconstitution of the polypeptides with a G protein beta subunit (G beta). The ability of recombinant G beta, produced in Sf9 cells, to assemble into stable beta gamma complexes (G beta gamma) with each of the G gamma processing intermediates was assessed. Both prenylated and unprenylated G gamma subunits formed stable complexes with G beta, but surprisingly, neither of the truncated-prenylated G gamma subunits were competent for this assembly. The G beta gamma complexes which were formed were examined for their ability to interact with a G protein alpha subunit (G alpha). Only those G beta gamma complexes containing a prenylated G gamma subunit were functional in this assay. These data indicate that: 1) prenylation of G gamma is not required for G beta gamma assembly; 2) assembly of the G beta gamma complex occurs prior to the proteolytic processing of G gamma; and 3) G beta gamma complexes require prenylated G gamma for interaction with G alpha.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

March 25, 1994

Volume

269

Issue

12

Start / End Page

9067 / 9073

Location

United States

Related Subject Headings

  • Trypsin
  • Recombinant Proteins
  • Protein Processing, Post-Translational
  • Protein Precursors
  • Protein Binding
  • Macromolecular Substances
  • GTP-Binding Proteins
  • Dimethylallyltranstransferase
  • Carboxypeptidases
  • Biochemistry & Molecular Biology
 

Citation

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MLA
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Higgins, J. B., and P. J. Casey. “In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.J Biol Chem 269, no. 12 (March 25, 1994): 9067–73.
Higgins, J. B., and P. J. Casey. “In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.J Biol Chem, vol. 269, no. 12, Mar. 1994, pp. 9067–73.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

March 25, 1994

Volume

269

Issue

12

Start / End Page

9067 / 9073

Location

United States

Related Subject Headings

  • Trypsin
  • Recombinant Proteins
  • Protein Processing, Post-Translational
  • Protein Precursors
  • Protein Binding
  • Macromolecular Substances
  • GTP-Binding Proteins
  • Dimethylallyltranstransferase
  • Carboxypeptidases
  • Biochemistry & Molecular Biology