THROMBIN RECEPTOR ACTIVATION RESULTS IN CALCIUM SIGNALING AND PROTEIN-KINASE C-DEPENDENT STIMULATION OF DNA-SYNTHESIS IN HEP-2G LARYNGEAL CARCINOMA-CELLS

BAGKGROUND. Recently, the expression of the ''tethered ligand'' thromb in receptor in carcinosarcoma and melanoma cells has been shown. Howev er, the role of the thrombin receptor in tumor cell metabolism still i s undefined. METHODS. In this article, the ''tethered ligand'' thrombi n receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antit hrombin receptor antibody and radioligand binding. Furthermore, the ef fects of cr-thrombin and thrombin receptor activating peptides (TRAP)- 6 on calcium mobilization, protein kinase C (PKC) translocation, and D NA synthesis were estimated, RESULTS. Pharmacologic characterization u sing [H-3]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [K-D] = 7.2 +/- 2.2 x 10 (-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The func tion of these binding sites was demonstrated by alpha-thrombin- and TR AP-6-induced mobilization of free intracellular calcium, and transloca tion of PKC from cytosol to cell membrane. Moreover, alpha-thrombin an d TRAP-6 induced an increase in [H-3]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide. CONCLUSIONS. To the authors' knowledge, the results of this study dem onstrate for the first time functional thrombin receptors in epidermoi d carcinoma cells. The thrombin receptor appears to be involved in gro wth regulation in HEp-2g cells by a PKC-dependent mechanism. (C) 1997 American Cancer Society.