Basic Research
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 15, 2003; 9(4): 800-803
Published online Apr 15, 2003. doi: 10.3748/wjg.v9.i4.800
Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase
Xing-Long Yang, Ya-Li Zhang, Zhuo-Sheng Lai, Fei-Yue Xing, Yu-Hu Liu
Xing-Long Yang, Ya-Li Zhang, Zhuo-Sheng Lai, Yu-Hu Liu, Institute of Gastroenterology, NanFang Hospital, First Military Medical University 510515, Guangzhou, Guangdong Province, China
Fei-Yue Xing, Key lab. for shock and microcirculation of PLA, Department of Preclinical Medicine, First Military Medical University 510515, Guangzhou, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Xing-Long Yang, Institute of Gastroenterology, NanFang Hospital, First Military Medical University 510515, Guangzhou, Guangdong Province, China. foru3000@hotmail.com
Telephone: +86-20-61364633 Fax: +86-20-61641544
Received: October 4, 2002
Revised: November 21, 2002
Accepted: November 28, 2002
Published online: April 15, 2003
Abstract

AIM: To study the detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC.

METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK2 residue 548 to 660 was amplified by PCR with the mRNA of human GRK2 (β1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK2 PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK2 PH domain fusion protein, BTK (Bruton’s tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK2 in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK2 PH domain, GST-GRK2 PH domain fusion protein and BTK PH domain to PKC in vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK2 to PKC was also detected by western blot and Co-immunoprecipitation.

RESULTS: The binding of GRK2 PH domain to PKC in vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK2 PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK2 PH domain.

CONCLUSION: GRK2 can bind with PKC and down-regulated PKC activity.

Keywords: $[Keywords]