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

FEBS Letters

Pagès, Gilles²  Pavirani, Andrea¹  Lecocq, Jean-Pierre¹  Rasmussen, Ulla B.¹  Pouysségur, Jacques²  Jallat, Sophie¹  Van Obberghen-Schilling, Ellen²  Schlesinger, Yasmin¹  Vouret-Craviari, Valérie² 

[1] Transgene, S.A., 11 rue de Molsheim, 67082 Strasbourg, France;Centre de Biochimie, C.N.R.S., Parc Valrose, 06034 Nice, France

关键词: α-Thrombin receptor;    G-protein;    Phospholipase C;    Polymerase chain reaction;    Oocyte expression;    Hamster fibroblast;   

DOI  :  10.1016/0014-5793(91)81017-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown