Al-Ani Bahjat, Hansen Kristina K, Hollenberg Morley D
Diabetes/Endocrine Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Mol Pharmacol. 2004 Jan;65(1):149-56. doi: 10.1124/mol.65.1.149.
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.
蛋白酶激活受体-2（PAR2）经胰蛋白酶切割后会暴露出一个拴系式受体激活序列S37LIGRLDTP。我们试图在PAR2拴系配体的氨基末端序列中确定负责受体激活的关键氨基酸残基。利用定点诱变技术，制备了9种在拴系配体前六个氨基酸（S37LIGRL42）中用丙氨酸替代的PAR2突变体：PAR2S37A、PAR2L38A、PAR2I39A、PAR2G40A、PAR2R41A、PAR2A37 - 38、PAR2A39 - 42、PAR2A37,39 - 42和PAR2A37 - 42，以及反向序列构建体PAR2L37S38。这些突变体与野生型PAR2（PAR2wt）一起在柯斯顿病毒转化的大鼠肾细胞中表达，然后在用胰蛋白酶和诸如SLIGRL - NH2等受体激活肽激活后，评估其受体介导的钙信号传导。此外，在上述细胞系中，使用定点抗受体抗体监测经胰蛋白酶激活后从PAR2切割下来的氨基末端受体序列的释放情况。所有PAR2构建体均被SL - NH2激活，且所有突变的拴系配体序列均被胰蛋白酶暴露。然而，观察到这些突变体中胰蛋白酶对受体的激活存在差异：PAR2突变体PAR2A37 - 38和PAR2L37S38中，氨基末端拴系配体的前两个残基（S37L38）要么变为丙氨酸，要么顺序颠倒，对胰蛋白酶几乎没有或没有反应，PAR2A37,39 - 42也是如此。然而，胰蛋白酶激活了所有其他构建体。我们得出结论，氨基末端拴系配体二肽序列S37L38在PAR2的激活中起主要作用。
