Abstract
The receptor responsible for the increase in intracellular calcium concentration ([Ca2+]i) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was observed with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin receptor is activated by thrombin cleavage to create a new NH2 terminus that acts as a tethered ligand, and peptides based on the tethered ligand can activate the receptor independently of thrombin. Northern analysis indicated the presence of mRNA encoding the platelet receptor in Saos-2 cells, and surface expression of the receptor was demonstrated by immunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single-letter amino acid code) was found to increase [Ca2+]i. The maximal response to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 +/- 6 microM peptide. The time course of the increase in [Ca2+]i with the peptide was different than that observed with thrombin; a pronounced shoulder was observed after an initial sharp rise. The phenylalanine in the second position of the agonist peptide and the arginine in the fifth position were shown to be essential for its activity. The requirement for proteolysis of the receptor for the thrombin-dependent increase in [Ca2+]i was demonstrated by two methods. Antibodies that reacted with the cleavage site of the receptor abolished the effect of thrombin on [Ca2+]i. In addition, a mutant of thrombin without catalytic activity as well as chemically inactivated thrombin failed to cause an increase in [Ca2+]i. Similar results were obtained with the rat osteoblast-like cell line UMR-106; a tethered ligand peptide based on the rat sequence induced an increase in [Ca2+]i, and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.
Highlights
Ter amino acid code) was found to increase[Ca2+]i.The The thrombin receptor responsible for platelet aggregation maximal response to the peptide was similar to that and secretion has recently been shown to be a member of the observed with thrombin, anda half-maximal response G-protein-coupled receptor family (Vu et al, 1991)
The results presented in this paper demonstrate that the [CaZ+lincrease in the osteosarcoma cell line Saos-2 in response to thrombin is due to the activation of a thrombin receptor similar to that found in platelets (Vu et al, 1991)
The newly created NHz terminus of the receptor acts as a tethereldigand, and peptides with the sequence of the tetheredligand can activate the receptor (Vu et al, 1991)
Summary
Ter amino acid code) was found to increase[Ca2+]i.The The thrombin receptor responsible for platelet aggregation maximal response to the peptide was similar to that and secretion has recently been shown to be a member of the observed with thrombin, anda half-maximal response G-protein-coupled receptor family (Vu et al, 1991). These results suggest that the response of Saos-2 patterns (data notshown), confirming that SFLL antibodies cells to thrombin depends on the cleavage of a receptor bind to a cell surface antigen under nonpermeabilized condi- homologous to the platelet receptor.
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