Fig. 7.

Role of Clathrin on UCN-II-Induced CRH-R2β Desensitization and Internalization in 293-R2β Cells

A, Clathrin membrane translocation after UCN-II treatment. HEK293 cells expressing CRH-R2β were stimulated with UCN-II (100 nm) for 2 min and CHC distribution was monitored over the ensuing time period by indirect immunofluorescence using specific primary antibodies and Alexa Fluor 488 secondary antibody (green). Identical results were obtained from four independent experiments. Also, CHC immunoreactivity was determined in membrane-rich fractions immunoblotted with antibodies against CHC (inset). Identical results were obtained from four independent experiments. B and C, Effect of CHC siRNA on UCN-II induced CRH-R2β desensitization (B) and internalization (C). 293-R2β Cells were transfected with 1 nmol CHC siRNA or scrambled oligonucleotide, and the effect on CHC protein expression was assessed by immunoblotting of total cell lysates with specific CHC or GAPDH antibodies. CRH-R2β homologous desensitization (induced by pretreatment with 100 nm UCN-II for 30 min) was also determined by measurement of UCN-II-induced cAMP production (B). Data represent the mean ± sem of two estimations from three independent experiments. *, P < 0.05 compared with cells without UCN-II pretreatment; +, P < 0.05 compared with control cells. Alternatively, internalization of UCN-II CRH-R2β was monitored by indirect confocal microscopy (C) using specific primary antibodies and Alexa Fluor 594 secondary antibody for CRH-R2β (red) and Alexa Fluor 488 secondary antibody for CHC (green). Identical results were obtained from four independent experiments. Scale bar, 20 μm. At least 20 individual cells in five random fields of view were examined. Inset, Fluorescence intensity of cytoplasmic CRH-R2β distribution from 20 individual cells in five random fields of view was examined, and the sum of fluorescence intensity of cytoplasmic (distance 4–18 μm) fluorescence was measured by the ImageJ software. Results are expressed as the mean ± sem of three estimations from 20 individual cells. *, P < 0.05 compared with control (scrambled sequence) cells.