cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization

FEBS Letters - Tập 288 - Trang 123-128 - 1991
Ulla B. Rasmussen1, Valérie Vouret-Craviari2, Sophie Jallat1, Yasmin Schlesinger1, Gilles Pagès2, Andrea Pavirani1, Jean-Pierre Lecocq1, Jacques Pouysségur2, Ellen Van Obberghen-Schilling2
1Transgene, S.A., 11 rue de Molsheim, 67082 Strasbourg, France
2Centre de Biochimie, C.N.R.S., Parc Valrose, 06034 Nice, France

Tóm tắt

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.

Tài liệu tham khảo

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FEBS Letters

Tập 288

123-128

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