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

Title:

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

Creator:

Matoušek, P., Novotný, J., and Svoboda, P.

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:b112ee99-344a-44db-a0ee-4d0b8eb49f86
uuid:b112ee99-344a-44db-a0ee-4d0b8eb49f86

Subject:

G-Proteins, Two-dimensional electrophoresis, Desensitization, Membrane domains, and Rafts

Type:

article, model:article, and TEXT

Description:

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.

Language:

English

Rights:

http://creativecommons.org/licenses/by-nc-sa/4.0/
policy:public

Source:

Physiological research | 2004 Volume:53 | Number:3

Harvested from:

CDK

Metadata only:

false