Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells

Abstract

Proteinase-activated receptors (PARs) can activate a number of signalling events, including T-cell signal-transduction pathways. Recent data suggest that the activation of PARs 1, 2 and 3 in Jurkat T-leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1. To activate the PARs, this study used the agonist peptides SFLLRNPNDK, SLIGKVDGTS and TFRGAPPNSF, which are based on the sequences of the tethered ligand sequences of human PARs 1, 2 and 3, respectively. Here, we show that peptides based on either the human or murine PAR(3)-derived tethered ligand sequences (TFRGAP-NH(2) or SFNGGP-NH(2)) do not activate PAR(3), but rather activate PARs 1 and 2, either in Jurkat or in other PAR-expressing cells. Furthermore, whilst thrombin activates only Jurkat PAR(1), trypsin activates both PARs 1 and 2 and also disarms Jurkat PAR(1) for thrombin activation. We conclude therefore that in Jurkat or related T cells, signalling via PARs that can affect VAV1 phosphorylation is mediated via PAR 1 or 2, or both, and that distinct serine proteinases may potentially differentially affect T-cell function in the settings of inflammation.

Figure 1

Calcium signalling in Jurkat cells by proteinase-activated receptor activating peptides (PAR-APs): concentration–effect curves. The calcium signal, relative to that obtained with A23187 (%A23187) was monitored at 530 nm (E₅₃₀) for increasing concentrations of the indicated PAR-AP. Symbols represent the mean value ± standard error of the mean (SEM, which is denoted by bars, except where error bars were smaller than the size of the symbols) for measurements performed with three or more cell aliquots. The data are representative of experiments carried out using two or more separately grown suspensions of Jurkat cells.

Figure 2

Desensitization of the TFRGAP-NH₂ (TFR-NH₂)-, SFNGGP-NH₂ (SFN-NH₂)-, and thrombin-mediated calcium signal by desensitization of proteinase-activated receptor 1 (PAR₁), proteinase-activated receptor 2 (PAR₂), or both. Jurkat cell suspensions were first exposed to receptor-desensitizing concentrations of either the PAR₁-activating peptide (PAR₁-AP), TFLLR-NH₂ (TFL-NH₂) (•, 100 µm, tracings a and d), the PAR₂-AP, SLIGRL-NH₂ (SL-NH₂) (▪, 200 µm, tracings b and e), or the PAR_1-2-AP, SFLLR-NH₂ (SFL-NH₂) (Δ, 200 µm, tracings c and f). After 5 min, a test concentration of TFR-NH₂ (▴, 500 µm), SFN-NH₂ (•, 500 or 1000 µm), or thrombin (Thr) (○, 5 U/ml), was added to the cell suspensions, with a continuous recording of calcium-mediated fluorescence (E₅₃₀, scale for time and fluorescence shown between tracings d and e). The calcium signal caused by TFR-NH₂ (▴, 500 µm), SFN-NH₂ (•, 500 or 1000 µm), or Thr (○, 5 U/ml), in a cell suspension that had not been previously exposed to TFL-NH₂ (tracings a and d), SL-NH₂ (tracings b and e), or SFL-NH₂ (tracings c and f), is shown to the right of each tracing. The data are representative of experiments carried out with three separately grown suspensions of Jurkat cells.

Figure 3

Calcium signalling in Kirsten virus-transformed rat kidney (KNRK) cells transfected with human proteinase-activated receptor 2 (PAR₂) (a) or rat PAR₂ (b): concentration–effect curves. The calcium signal, relative to that obtained with A23187 (%A23187), was monitored at 530 nm (E₅₃₀) for increasing concentrations of the indicated PAR-activating peptide (PAR-AP). Symbols represent the mean value ± standard error of the mean (SEM, which is denoted by bars, except where error bars were smaller than the size of the symbols) for measurements carried out with three or more cell aliquots. The data are representative of experiments carried out with two or more separately grown monolayers of KNRK cells.

Figure 4

Desensitization of the thrombin (Thr)-, and trypsin (Trp)-mediated calcium signal by desensitization of proteinase-activated receptor 1 (PAR₁) or proteinase-activated receptor 2 (PAR₂). Jurkat cell suspensions were first exposed to receptor-desensitizing concentrations of either the PAR₁-AP, TFLLR-NH₂ (TFL-NH₂) (•, 100 µm, tracings a and b) or the PAR₂-AP, SLIGRL-NH₂ (SL-NH₂) (▪, 200 µm, tracings c and d). After 5 min, a test concentration of Thr (○, 5 U/ml) or Trp (□, 25 U/ml) was added to the cell suspensions, with a continuous recording of calcium-mediated fluorescence (E₅₃₀, scale for time and fluorescence shown between tracings b and c). The calcium signal caused by Thr (○, 5 U/ml) or Trp (□ 25 U/ml), in a cell suspension that had not been previously exposed to either TFL-NH₂ (tracings a and b) or SL-NH₂ (tracings c and d), is shown to the right of each tracing. The data are representative of experiments carried out with three separately grown suspensions of Jurkat cells.

Figure 5

Concentration–desensitization (•, ○) and concentration–stimulation (□) curves for trypsin-mediated activation of Jurkat cells. Jurkat cell suspensions were first stimulated by the addition of trypsin (2–200 nm; 1–100 U/ml), and the calcium signal (%A23187, white square), relative to that obtained with 2 µm A23187, was measured. Following trypsin treatment, cells were exposed to either 25 µm TFLLR-NH₂ (•) or 50 nm (5 U/ml) thrombin (○) to monitor proteinase-activated receptor 1 (PAR₁) activation. The residual fluorescence signal measured after trypsin pretreatment was expressed as a percentage [% residual PAR₁ stimulation monitored by TFLLR-NH₂ (•) or thrombin (○)] of the control fluorescence signal measured without earlier trypsin treatment, by activating PAR₁ with either TFLLR-NH₂ (•) or thrombin (○). Symbols represent the mean value ± standard error of the mean (SEM, which is denoted by bars, except where error bars were smaller than the size of the symbols) for measurements carried out with three or more cell aliquots. The data are representative of experiments performed with two or more separately grown suspensions of Jurkat cells.