title: Proteinase-activated receptor-2: key role of amino-terminal dipeptide residues of the tethered ligand for receptor activation.
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, PAR2A37-38, PAR2A39-42, PAR2A37,39-42, and PAR2A37-42, 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 PAR2A37-38 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 PAR2A37,39-42. 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.
authors.affiliation: Diabetes/Endocrine Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Bahjat
authors.initials: B
authors.lastname: Al-Ani
authors.forename: Kristina K
authors.initials: KK
authors.lastname: Hansen
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D004151:Dipeptides / Q000737:chemistry / Q000494:pharmacology*; D008024:Ligands; D051381:Rats; D044464:Receptor, PAR-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D008024:Ligands; D044464:Receptor, PAR-2; D014357:Trypsin