The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.
A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).
Department of Medicine, State University of New York, Stony Brook 11794.
W F
WF
Bahou
B S
BS
Coller
C L
CL
Potter
K J
KJ
Norton
J L
JL
Kutok
M S
MS
Goligorsky
D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000648:ultrastructure; D016466:CHO Cells / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000648:ultrastructure; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000502:physiology; D010974:Platelet Aggregation / Q000276:immunology; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000378:metabolism; D014471:Umbilical Veins
D007074:Immunoglobulin G; D008024:Ligands; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D002118:Calcium