CHARACTERIZATION OF THE RECEPTOR RESPONSIBLE FOR THROMBIN-INDUCED INTRACELLULAR CALCIUM RESPONSES IN OSTEOBLAST-LIKE CELLS

The receptor responsible for the increase in intracellular calcium con centration ([Ca2+]i) after the addition of thrombin to the human osteo blast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was obs erved with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin recept or 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 i mmunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single- letter amino acid code) was found to increase [Ca2+]i. The maximal res ponse to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 +/- 6 muM peptide. The tim e course of the increase in [Ca2+]i with the peptide was different tha n that observed with thrombin; a pronounced shoulder was observed afte r an initial sharp rise. The phenylalanine in the second position of t he agonist peptide and the arginine in the fifth position were shown t o be essential for its activity. The requirement for proteolysis of th e receptor for the thrombin-dependent increase in [Ca2+]i was demonstr ated 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 chemicall y 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 increa se in [Ca2+]i, and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.