Physiol. Res. 53: 295-303, 2004

Resolution of Gsα and Gqα/G11α Proteins in Membrane Domains by Two-Dimensional Electrophoresis: The Effect of Long-Term Agonist Stimulation


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

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

© 2004 by the Institute of Physiology, Czech Academy of Sciences