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 effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K(D) = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I-ATAP2 and 125I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (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, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.
Weinstein
J R
JR
Department of Microbiology and Molecular Genetics, University of California, Irvine 92697, USA.
Lau
A L
AL
Brass
L F
LF
Cunningham
D D
DD
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D007003:Hypoglycemia / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D009417:Nerve Tissue / Q000293:injuries*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018179:Receptors, Thrombin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014947:Wounds and Injuries / Q000503:physiopathology*
D000911:Antibodies, Monoclonal; D008246:Lysophospholipids; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate