cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization.
The serine protease alpha-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.
Rasmussen
U B
UB
Transgene, S.A., Strasbourg, France.
Vouret-Craviari
V
V
Jallat
S
S
Schlesinger
Y
Y
Pagès
G
G
Pavirani
A
A
Lecocq
J P
JP
Pouysségur
J
J
Van Obberghen-Schilling
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000235:genetics*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D010738:Type C Phospholipases / Q000235:genetics*; D014981:Xenopus
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium