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

Abstract

1. 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. 2. 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. 3. 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. 4. 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. 5. We conclude that the acidic EL-2 tripeptide, PEE, in PAR2 plays an important role in governing agonist activity. 6. 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. 7 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.

Figure 1

Sequences of PAR₂ variants compared with PAR₂ and PAR₁ (upper panel) and their expression in KNRK cells (middle and lower panel). (Upper panel) The sequences of the extracellular loop 2 of wtPAR₂ (PAR₂) and the two receptor variants (PAR₂PRR, PAR₂NET) are compared with the extracellular loop 2 sequence of rat PAR₁. The bold print of the PAR₁ sequence denotes the region of high sequence homology between PAR₁ and PAR₂. (Middle panel) Cellular fluorescence intensity detected with the B5 anti-PAR₂ antiserum was monitored by fluorescence-activated cell sorting for untransfected KNRK cells (dashed lines; same signal was observed for vector-transfected cells) or for wt-PAR₂-expressing cells (solid lines). (Lower panel) The expression of receptor mRNAs for the host wild-type KNRK cells, (Wt-PAR₂), PAR₂PRR (PRR) and PAR₂NET (NET) were determined by RT–PCR. The RT–PCR signal observed for actin was the same in all cell lines (not shown). The positions of the oligonucleotide size markers (in base-pairs) are shown on the left (lane M). The position of the expected PCR product for PAR₂ (560 base-pairs) is shown on the right. Identical amounts of RNA obtained from each cell line were subjected to analysis by RT–PCR, as outlined in Methods.

Figure 2

Calcium signalling by wt-PAR₂ (a, b), PAR₂PRR (c, d) and PAR₂NET (e, f). Comparative responses to SLIGRL-NH₂ (SL-NH₂, ○) and SLIGEL-NH₂ (SE-NH₂, ▪) [fluorescence (E₅₃₀), reflecting increases in intracellular calcium] were monitored in fluo-3-loaded cell lines stimulated by either SLIGRL-NH₂ (a, c, e) or SLIGEL-NH₂ (b, d, f). The concentrations of peptide agonists were adjusted in an attempt to show comparable increases in fluorescence, relative to the signal yielded in each cell sample by 2 μM of the ionophore A23187 (▴). (a, b) wt-PAR₂; (c, d) PAR₂PRR; (e, f) PAR₂NET. The PAR₂NET cell line responded poorly to relatively high concentrations of SLIGEL-NH₂ (f and Figure 5).

Figure 3

Concentration-effect curves for PAR-APs and trypsin in wt-PAR₂ cells. The fluorescence responses relative to the signal caused in identical cell suspensions by 2 μM A23187 (E₅₃₀: %A23187) were measured in replicate cell suspensions, as outlined in Methods, for increasing concentrations of the indicated PAR-APs and trypsin. Values represent the averages (±s.e.mean, bars) for measurements done with four or more replicate cell suspensions coming from two or more independently grown crops of cells. Error bars smaller than the symbols are not shown.

Figure 4

Concentration effect curves for PAR-APs and trypsin in PAR₂PRR cells. The fluorescence responses relative to the signal caused by 2 μM A23187 (E₅₃₀: %A23187) were measured for increasing concentrations of the indicated PAR-APs and trypsin, exactly as described in the legend to Figure 3.

Figure 5

Concentration-effect curves for PAR-APs and trypsin in PAR₂-NET cells. Concentration-effect curves for the fluorescence response relative to 2 μM A23187 (E₅₃₀: %A23187) were obtained for the same PAR-APs and trypsin, as outlined in the legends to Figures 3 and 4.