Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase

Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obliga tory role in regulating the activity of, endothelial nitric oxide (NO) synt hase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uter ine vasodilatation during pregnancy and estrogen replacement therapy. To te st this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental arte ry endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Res triction digestion of these clones revealed that the cDNAs of oCav-1 ranged from similar to 2.1 to 2.7 kb. Not-them analysis of Cav-1 mRNAs in ovine u terine artery endothelial cells (UAEC) showed two transcripts of similar to 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveol in-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that i n addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a similar to 1.6-2.1 k b 3'-untranslated region. Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidin e residues were tagged at the end of its COOH-terminus and developed a [His ](6)-tagged oCav-1 'pull-down assay' for studying the association of eNOS w ith Cav-1. Incubation of exogenous [His](6)-tagged oCav-1 with resting UAEC extracts led to the formation of a [His](6)-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) p eptide, but not a mutated CSD peptide, [His](6)-lagged oCav-1 associated eN OS was dose (0-10 muM)-dependently inhibited, eNOS association with Cav-1 i n UAEC was further confirmed by the facts that eNOS co-immunoprecipitated w ith Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the is olation of caveolae membranes. Because dissociation of eNOS from Cav-1 is r equired for the activation of eNOS, eNOS association with Cav-1 in UAEC sug gests an important role of Cav-1 in regulating UAEC production of NO and po ssibly NO-mediated uterine vasodilatation. (C) 2001 Elsevier Science Irelan d Ltd. All rights reserved.