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 obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test 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 artery endothelial cells. Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb. Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 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 histidine 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 with 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) peptide, but not a mutated CSD peptide, [His]6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited. eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes. Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation.
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Perinatal Research Laboratories, Department of Obstetrics and Gynecology, University of Wisconsin-Madison, 7E. Meriter Hospital, 202 S. Park Street, Madison, WI 53715, USA. dongbaochen@facstaff.wisc.edu
Zangl
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Zhao
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Markley
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Zheng
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Bird
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Magness
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D000818:Animals; D001158:Arteries; D001483:Base Sequence; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D004926:Escherichia coli / Q000502:physiology*; D005260:Female; D023421:Models, Animal; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D011247:Pregnancy; D011485:Protein Binding; D012756:Sheep; D014599:Uterus / Q000098:blood supply
D051242:Caveolin 1; D022461:Caveolins; D018076:DNA, Complementary; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III