Resolution of Gsα and Gqα/G11α
Proteins in Membrane Domains by Two-Dimensional Electrophoresis: The
Effect of Long-Term Agonist Stimulation
P. MATOUŠEK1,2, J. NOVOTNÝ1,2, P. SVOBODA1,2
1Institute of Physiology, Academy of Sciences of the Czech
Republic, 2Laboratory of Molecular Pharmacology, Department of
Physiology, Faculty of Natural Sciences, Charles University, Prague, Czech
Republic
Received April 23, 2003
Accepted August 7, 2003
Summary
Low-density membrane-domain fractions were prepared from S49 lymphoma
cells and clone e2m11 of HEK293 cells expressing a large number of
thyrotropin-releasing hormone receptor (TRH-R) and G11 by
flotation on sucrose density gradients. The intact cell structure was
broken by detergent-extraction, alkaline-treatment or drastic
homogenization. Three types of low-density membranes were resolved by
two-dimensional electrophoresis and analyzed for Gsα (S49) or Gqα/G11
(e2m11) content. Four individual immunoblot signals of Gsα
protein were identified in S49 lymphoma cells indicating complete
resolution of the long GsαL±ser and
short GsαS±ser variants of Gsα.
All these were diminished by prolonged agonist (isoprenaline) stimulation.
In e2m11-HEK cells, five different immunoblot signals were detected
indicating post-translational modification of G proteins of Gqα/G11α
family. The two major spots corresponding to exogenously (over)expressed G11α
and endogenous Gqα were reduced; the minor spots diminished by hormonal
stimulation. Parallel analysis by silver staining of the total protein
content indicated that no major changes in protein composition occurred
under these conditions. Our data thus indicate that agonist-stimulation of
target cells results in down-regulation of all different members of Gs
and Gq/G11 families. This agonist-specific effect
may be demonstrated in crude membrane as well as domain/raft preparations
and it is not accompanied by changes in overall protein composition.
Key words
G-Proteins • Two-dimensional electrophoresis • Desensitization • Membrane
domains • Rafts
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