Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains.
Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) gamma1 to the rafts. Depletion of cholesterol by methyl-beta-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCgamma1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.
Hur
Eun-Mi
EM
Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784, Republic of Korea.
Park
Yong-Soo
YS
Lee
Byoung Dae
BD
Jang
Il Ho
IH
Kim
Hyeon Soo
HS
Kim
Tae-Don
TD
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
Kim
Kyong-Tai
KT
D000818:Animals; D001920:Bradykinin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D015151:Immunoblotting; D008055:Lipids / Q000737:chemistry*; D021962:Membrane Microdomains; D008954:Models, Biological; D016716:PC12 Cells; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D008055:Lipids; D004815:Epidermal Growth Factor; D002784:Cholesterol; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D001920:Bradykinin; D002118:Calcium