Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells

Tetsuzo Nakayama, Katsuya Hirano, Mayumi Hirano, Junji Nishimura, Hirotaka Kuga, Katsuya Nakamura, Shosuke Takahashi, Hideo Kanaide

研究成果: ジャーナルへの寄稿記事

9 引用 (Scopus)

抄録

Proteolysis plays an important role in inactivating protease-activated receptor-1 (PAR1). We aimed to determine the cleavage site(s) responsive for the proteolytic inactivation of PAR1 in human umbilical vein endothelial cells. Fura-2 fluorometry revealed that the preceding stimulation with trypsin abolished the subsequent [Ca 2+ ] i response to thrombin, while the responses to PAR1-activating peptides remained intact. On the other hand, thrombin had no effect on the subsequent response to trypsin. The immunostaining with antibodies against the residues 35-46 (SPAN12) and 51-64 (WEDE15) revealed the broad boundaries of cleavage. Trypsin removed both epitopes from the cell surface within 3 min, while thrombin removed the epitope of SPAN12. The longer incubation with thrombin removed the epitope of WEDE15. However, PAR1-activating peptides thereafter induced an attenuated but significant elevation of [Ca 2+ ] i . Not only the receptor internalization as observed with a confocal microscope, but also an additional cleavage was thus suggested to contribute to the thrombin-induced removal of the epitope of WEDE15. The analyses of the PAR1 mutants identified three cleavage sites for trypsin; residues 41-42, 70-71 and 82-83. The cleavage at the latter two sites was suggested to dominate that at the former, and thus remove the ligand region (residues 42-47). The inactivation of PAR1 due to proteolytic removal of the ligand region may contribute not only to the inactivation of PAR1 by proteases such as trypsin, but also to the termination of the intracellular signaling initiated by thrombin in the vascular endothelial cells.

元の言語英語
ページ(範囲)23-32
ページ数10
ジャーナルBiochemical Pharmacology
68
発行部数1
DOI
出版物ステータス出版済み - 7 1 2004

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PAR-1 Receptor
Endothelial cells
Thrombin
Endothelial Cells
Ligands
Trypsin
Epitopes
Proteolysis
Fluorometry
Peptides
Fura-2
Human Umbilical Vein Endothelial Cells
Microscopes
Peptide Hydrolases
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

これを引用

Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells. / Nakayama, Tetsuzo; Hirano, Katsuya; Hirano, Mayumi; Nishimura, Junji; Kuga, Hirotaka; Nakamura, Katsuya; Takahashi, Shosuke; Kanaide, Hideo.

:: Biochemical Pharmacology, 巻 68, 番号 1, 01.07.2004, p. 23-32.

研究成果: ジャーナルへの寄稿記事

Nakayama, Tetsuzo ; Hirano, Katsuya ; Hirano, Mayumi ; Nishimura, Junji ; Kuga, Hirotaka ; Nakamura, Katsuya ; Takahashi, Shosuke ; Kanaide, Hideo. / Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells. :: Biochemical Pharmacology. 2004 ; 巻 68, 番号 1. pp. 23-32.
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