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

Abstract

The scrine protease a-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung Abroblasts. To clone a thrombin receptor cDNA, selcctivc amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerasc chain reaction. Using rcverae 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 oocytcs leads to the expression of a functional thrombin receptor. The hamster thrornbin receptor consists of 427 amino acid residues with 8 hydrophobic domains, in&ding 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 squcnce followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exositc of thrombin.u-Thrombin receptor; G-protein; Phospholipase C; Polymerase chain reaction; Oocyte expression; Hamster ftbroblast

Characterization of a new peptide agonist of the protease-activated receptor-1

“…However, physiologically these peptides do not exist and the PARs are activated by proteases. Upon proteolytic activation by thrombin, the tethered ligand intramolecularly activates the receptor [10,11]. Thus, the concentration of the tethered ligand should be the same physiologically.…”

“…Thus it appears that the Leu 44 could be substituted with Arg residue and hence is not essential for the activity of the peptide. It is interesting that the corresponding residue to Leu 44 in murine and hamster PAR1 is Phe [10,11,[43][44][45]. In addition, mutation of this residue to a proline did not affect the activity of the peptide [46].…”

“…Earlier work from Brass and co-workers has identified these two residues as the most important in the PAR-1 activating peptides [43]. Among various species, these two residues (Phe and Arg) are highly conserved in the PAR-1 [10,11,44,45,47]. However, in the model proposed by Seeley et al, the R 46 (fifth residue in the peptide) does not interact with the LBS-1 [41].…”

“…Thrombin activates the PARs through an unique mechanism involving proteolytic cleavage of the amino terminal of the receptor and thus exposing a new amino terminus. The newly formed amino terminus binds to the extracellular domain of the receptor to cause intracellular signaling [10,11]. Numerous studies on the structure-function analysis of PAR1 agonists have revealed a requirement of free amine group at the amino terminus, a short chain amino acid at position 1, aromatic amino acid at position 2 and arginine at position 5 for potent activation of PAR-1 [12,13].…”

von Hippel‐Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation

“…The activation of PAR1, the prototype of this family, requires proteolytic cleavage of an Arg-Ser bond in the NH 2 -terminal exodomain by serine proteases that generates a new NH 2 -terminal that functions as a tethered ligand. 14,18 Recent studies have reported that high levels of PAR1 are present in several cancer cells where it plays a important role in cancer cell proliferation and motility. 17,19,20 Several studies have shown that morphological changes in actin organization induced by thrombin are mediated by PAR signaling through RhoA activation in endothelial cells, [21][22][23] but only a few studies have examined this process in cancer cells.…”