Abstract

Tryptic cleavage of proteinase-activated receptor-2 (PAR₂) causes the unmasking of a tethered receptor-activating sequence, S³⁷LIGRLDTP.... We sought to determine, in the amino-terminal sequence of the PAR₂ tethered ligand, the key amino acid residues that are responsible for receptor activation. Using site-directed mutagenesis, nine PAR₂ mutants with alanine substitutions in the first six amino acids of the tethered ligand, S³⁷LIGRL⁴²..., were prepared: PAR₂S³⁷A, PAR₂L³⁸A, PAR₂I³⁹A, PAR₂G⁴⁰A, PAR₂R⁴¹A, PAR₂A^37-38, PAR₂A^39-42, PAR₂A^37,39-42, and PAR₂A^37-42, along with the reverse-sequence construct, PAR₂L³⁷S³⁸. These mutants, together with wild-type PAR₂(PAR₂wt), were expressed in Kirsten virus-transformed rat kidney cells and were then assessed for receptor-mediated calcium signaling upon activation by trypsin and by receptor-activating peptides like SLIGRL-NH₂. In addition, the release of the N-terminal receptor sequence that is cleaved from PAR₂ by trypsin activation was monitored in the above cell lines using a site-targeted anti-receptor antibody. All PAR₂ constructs were activated by SL-NH₂, and all mutated tethered ligand sequences were unmasked by trypsin. However, differential activation of the receptor by trypsin in these mutants was observed: PAR₂ mutants PAR₂A^37-38 and PAR₂L³⁷S³⁸, in which the first two amino-terminal tethered ligand residues (S³⁷L³⁸) are either changed to alanines or reversed, yielded little or no response to trypsin, nor did PAR₂A^37,39-42. However, trypsin activated all other constructs. We conclude that the amino-terminal tethered ligand dipeptide sequence S³⁷L³⁸ plays a major role in the activation of PAR₂.