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
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|