Proteinase activated receptor 2: role of extracellular loop 2 for ligand-mediated activation

Authors

  • Bahjat Al-Ani,

    1. Endocrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
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  • Mahmoud Saifeddine,

    1. Endocrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
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  • Atsufumi Kawabata,

    1. Endocrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
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    • 4

      Current address: Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577 Japan

  • Morley D Hollenberg

    Corresponding author
    1. Endocrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
    2. Department of Pharmacology & Therapeutics, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
    3. Department of Medicine, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1
      Endorcrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1; E-mail: mhollenb@acs.ucalgary.ca
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Endorcrine Research Group, University of Calgary, Faculty of Medicine, Calgary, AB, Canada T2N 4N1; E-mail: mhollenb@acs.ucalgary.ca

Abstract

  • Rat proteinase-activated receptor-2 (PAR2) variants were stably expressed in rat KNRK cells: (a) wild-type (wt)–PAR2; (b) PAR2PRR, with the extracellular loop 2 (EL-2) sequence P231E232E233mutated to PRR and (c) PAR2NET, with the EL-2 sequence, PEEV changed to NETL. Cell lines were evaluated for their sensitivity (calcium signalling) towards trypsin and the receptor-activating peptides, SLIGRL-NH2, SLIGEL-NH2, trans-cinnamoyl(tc)-LIGRLO-NH2, and SFLLR-NH2.

  • SLIGEL-NH2 exhibited low potency (1 : 200 relative to SLIGRL-NH2) in wild-type PAR2. Its activity was increased 5 fold in PAR2PRR, but it was inactive in PAR2NET.

  • In PAR2PRR, the potencies of SLIGRL-NH2, tc-LIGRLO-NH2, and SFLLR-NH2 were decreased by 80–100 fold. But, the potency of trypsin was decreased by only 7 fold.

  • In PAR2NET, highly homologous in EL-2 with proteinase-activated receptor-1 (PAR1), the potency of the PAR1-derived peptide, SFLLR-NH2, was reduced by 100 fold compared with wt-PAR2, whereas the potency of the PAR2-derived AP, SLIGRL-NH2 was reduced 10 fold. In contrast, the potency of trypsin in PAR2NET was almost the same as in wt-PAR2.

  • We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity.

  • The data obtained with the PEEV→NETL mutation suggested: (a) that SLIGRL-NH2 and SFLLR-NH2 interact in a distinct manner with PAR2 and (b) that SFLLR-NH2 may interact differently with PAR2 than it does with PAR1.

  • The differential reductions in the potencies of SLIGRL-NH2, compared with trypsin in the PAR2PRR and PAR2NET cell lines point to differences between the interactions of the trypsin-revealed tethered ligand and the free receptor-activating peptide with PAR2.

British Journal of Pharmacology (1999) 128, 1105–1113; doi:10.1038/sj.bjp.0702834

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