Fig. 11.

Role of CRH-R2β C-Terminal Amino Acid Cassette TAAV on Receptor Desensitization and β-Arrestin Recruitment

A, Receptor (wild-type or mutant) expression was monitored by indirect fluorescent confocal microscopy using specific primary antibodies and Alexa Fluor 594 secondary antibodies for CRH-R2β (red). Identical results were obtained from three independent transfections. B, Alternatively, cells were pretreated with UCN-II (100 nm) for 30 min to induce desensitization. After extensive washing, cAMP response to a second UCN-II stimulus (100 nm for 15 min) was determined. Data represent the mean ± sem of two estimations from three independent experiments. *, P < 0.05 compared with wild type CRH-R2 cAMP response. C, To identify mutant CRH-R2β and β-arrestin complex formation, 293-R2β cells transiently expressing wild-type or mutant CRH-R2β were stimulated with UCN-II (100 nm) for 2 min before solubilization of membrane-rich fractions and immunoprecipitation with specific CRH-R1/2 antibodies. Proteins were resolved on SDS-PAGE gels, followed by immunoblotting with pan-arrestin (bottom panel) antibodies to identify potential complex formation. Representative immunoblots are presented. Identical results were obtained from four independent experiments. Quantification was carried out by densitometry scanning. Data represent the mean ± sem of two estimations from three independent experiments. *, P < 0.05 compared with basal.