HEK293細胞中細絲蛋白-C對α(下標 1A)-腎上腺素受體介導細胞外信號調節激酶磷酸化作用的影響

AIM To find novel non-G-proteins that may interact with the α(subscript 1A)-adrenergic receptor (α(subscript 1A)-AR) and study the effects of the proteins on receptor cell signaling. METHODS Yeast two-hybrid assay was performed, using the C terminal tail of α(subscript 1A)-AR (α(subscript 1A)-AR-CT) as a bait protein, to screen human brain cDNA library. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and o-nitropheny1 β-D-galactopyranoside (ONPG) assay were subsequently carried out to confirm the positive interactions. Transfection with lipofactamine and immunoblotting assay were used to observe the effects of filamin-C (FLN C) on α(subscript 1A)-AR cell signaling. RESULTS ① The C terminal part of FLN C was found to interact with α(subscript 1A)-AR-CT on selection medium in yeast. ② In X-Gal assay, co-transformants of FLN C and α(subscript 1A)-AR-CT turned blue at 6 h, while control yeast clone did not; The results of ONPG assay indicated the β galactopyranoside activity in co-transformants were about 2-3 fold higher than control (P＜0.01). ③ Level of extracellular signal-regulated kinase (ERK1/2) phosphorylation induced by phenylephrine (10μmol•L^(-1), 30 min) increased greatly when C terminal part of FLN C protein was expressed instantly in the human embryonic kidney 293 (HEK293) cells expressing full-length α(subscript 1A)-AR stably. CONCLUSION Fragments of FLN C could bind to α(subscript 1A)-AR-CT in yeast cells and enhance the level of ERK1/2 phophorylation induced by α(subscript 1A)-AR in HEK293 cells.