Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients.
We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of 125I-labeled TRP-14 was found to be saturable with a affinity constant of 2 microM and maximum binding of 41 pmol/mg of cell protein. The 125I-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of 125I-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (> 1 microM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Tiruppathi
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C
Department of Physiology and Cell Biology, Albany Medical College, New York 12208.
Lum
H
H
Andersen
T T
TT
Fenton
J W
JW
Malik
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AB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin; C027962:diisopropylphosphoryl-thrombin; D002118:Calcium