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Research ArticleArticle

Serotonin-Mediated Palmitoylation and Depalmitoylation of GAlpha Proteins in Rat Brain Cortical Membranes

Shubhada Bhamre, Hoau-Yan Wang and Eitan Friedman
Journal of Pharmacology and Experimental Therapeutics September 1998, 286 (3) 1482-1489;
Shubhada Bhamre
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Hoau-Yan Wang
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Eitan Friedman
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Abstract

We investigated serotonin stimulated palmitoylation of Galpha subunits in rat brain cerebrocortical membranes. Serotonin dose dependently stimulated palmitoylation of membrane Galpha proteins. The highest [3H] palmitate incorporation observed was by G alpha-q (7-fold), followed by G alpha-o (5-fold), G alpha-i (4-fold) and G alpha-s (3-fold) and these increases in palmitoylation were blocked by methiothipin, a serotonin receptor antagonist. Isoproterenol selectively stimulated Galpha-s palmitoylation which was blocked by propranalol. Immunoprecipitates of palmitoylated G alpha subunits yielded single labeled bands on SDS-PAGE. In an attempt to define the sequence of palmitoylation/depalmitoylation that follows receptor stimulation, nonreceptor mediated palmitoylation was carried out in the presence of guanine nucleotides and receptor mediated Galpha depalmitoylation was then monitored. Receptor stimulation did not result in depalmitoylation when membranes were prelabeled with [3H] palmitic acid in the presence of the nonhydrolyzable analogue of GTP, Gpp(NH)p. However, serotonin receptor stimulation in the presence of guanine nucleotides, depalmitoylated (90%) membrane G alpha proteins when prelabeled in the presence of GTP. Coimmunoprecipitation experiments revealed decrease in G beta immunoreactivity associated with Galpha immunoprecipitates obtained from membranes prelabeled in presence of GTP prior to reincubation with Gpp(NH)p and serotonin. These observations suggest that receptor occupation results in depalmitoylation of the trimer, followed by guanine nucleotide exchange and dissociation of the alpha subunit frombeta-gamma dimer and that the activatedalpha subunit is a substrate for repalmitoylation.

Footnotes

  • Send reprint requests to: Dr. E. Friedman, Department of Pharmacology, MCP - Hahnemann School of Medicine, Allegheny University of the Health Sciences, 3200 Henry Avenue, Philadelphia, PA 19129. E-mail: Friedmane{at}auhs.edu

  • ↵1 This study was supported by United States Public Health Service Grants AG07700 and NS29514.

  • Abbreviations:
    HEPES
    4-(2-hydroxyethyl)-1-piperazineetanesulfonic acid
    EG TA
    ethylene glycol bis (beta-aminoethyl N, N,N′,N′-tetraacetic acid)
    EDTA
    ethylenediaminetetraacetic acid
    PMSF
    phenylmethyl sulfonyl fluoride
    SDS
    sodium dodecyl sulfate
    TBS
    Tween-20 containing phosphate-buffered saline
    • Received January 22, 1998.
    • Accepted April 15, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 286, Issue 3
1 Sep 1998
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Research ArticleArticle

Serotonin-Mediated Palmitoylation and Depalmitoylation of GAlpha Proteins in Rat Brain Cortical Membranes

Shubhada Bhamre, Hoau-Yan Wang and Eitan Friedman
Journal of Pharmacology and Experimental Therapeutics September 1, 1998, 286 (3) 1482-1489;

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Research ArticleArticle

Serotonin-Mediated Palmitoylation and Depalmitoylation of GAlpha Proteins in Rat Brain Cortical Membranes

Shubhada Bhamre, Hoau-Yan Wang and Eitan Friedman
Journal of Pharmacology and Experimental Therapeutics September 1, 1998, 286 (3) 1482-1489;
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