Localization of VEGFR-2 and PL[D.sub.2] in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK

To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase [D.sub.2] (PL[D.sub.2]), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-[delta] regulates the VEGF-induced activation of PLD/ERK. Thus PL[D.sub.2] could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-[delta] pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-[beta]-cyclodextrin (M[beta]CD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to M[beta]CD-treated ceils restored caveolar structures. Pretreatment with M[beta]CD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade. caveolin-1; protein kinase C-[delta]; signaling; vascular endothelial growth factor; phospholipase D