INJURY-RELATED FACTORS AND CONDITIONS DOWN-REGULATE THE THROMBIN RECEPTOR (PAR-1) IN A HUMAN NEURONAL CELL-LINE

Previous studies have demonstrated that thrombin can induce potent eff ects on neural cell morphology, biochemistry, and viability. Nearly ai l of these effects are mediated by proteolytic activation of the throm bin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneu ral cell types have been shown to be novel and cell type specific; how ever, little is known about PAR-1 regulation in neural cells. In the p resent study, PAR-1 cell surface expression and regulation were examin ed in a transformed retinoblast (Ad12 HER 10) cell line using radioiod inated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact f orm of the receptor. Scatchard analysis revealed high-affinity, specif ic binding to a single affinity class of receptors: K-D = 3.13 and 5.2 5 nM, B-max = 190.1 and 67.8 fmol/mg of protein for I-125-ATAP2 and I- 125-SPAN12, respectively. Specificity for PAR-I was confirmed by demon strating rapid and near complete decreases for both antibodies followi ng treatment with thrombin or PAR-1 activating peptide (SFLLRN). Diffe rential antibody binding was used to demonstrate rapid and near comple te thrombin-induced PAR-1 cleavage and internalization, with protein s ynthesis-dependent replacement of intact receptors occurring over long er time intervals, but only minimal recycling of cleaved receptors, A variety of factors and conditions were screened for their effects on P AR-1 expression. Significant decreases in PAR-1 expression were induce d by the protein kinase C activator phorbol 12-myristate 13-acetate (8 7% at 3 h), the phospholipid inflammatory mediator lysophosphatidic ac id (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, res pectively). The effect of hypoglycemia was shown by RNase protection t o be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally wit h PAR-1 cell surface expression.