Kahn M, Ishii K, Kuo W L, Piper M, Connolly A, Shi Y P, Wu R, Lin C C, Coughlin S R
Department of Medicine, University of California, San Francisco 94143-0524, USA.
Mol Med. 1996 May;2(3):349-57.
Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes.
The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes.
Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating these two genes is less than 100 kb.
The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event. These results define a new protease-activated receptor gene cluster in which new family members may be found.
凝血酶是一种能引发多种细胞反应的丝氨酸蛋白酶。凝血酶受体的分子克隆揭示了一种通过新型蛋白水解机制激活的G蛋白偶联受体。最近，发现了第二种蛋白酶激活受体，并将其命名为PAR2。PAR2在序列上与凝血酶受体高度相关，并且与凝血酶受体一样，通过其氨基末端胞外域的切割而被激活。同样与凝血酶受体一样，PAR2可被与其拴系配体序列相对应的六肽激活，而与受体切割无关。因此，在功能上，凝血酶受体和PAR2构成了一个具有新型蛋白水解激活机制的新兴受体家族。为了进一步探索两种已知蛋白酶激活受体的相关性，并研究是否可能存在蛋白酶激活基因簇，我们比较了凝血酶受体和PAR2基因的结构和染色体定位。
通过分析λ噬菌体、P1噬菌体和细菌人工染色体（BAC）基因组克隆来确定两种蛋白酶激活受体基因的基因组结构。用中期染色体上的荧光原位杂交（FISH）确定染色体定位，并通过双色FISH以及对包含这两个基因的酵母人工染色体（YAC）和BAC进行分析来评估这两个基因之间的相对距离。
对基因组克隆的分析表明，两种蛋白酶激活受体基因具有两外显子的基因组结构，其中第一个外显子编码5'非翻译序列和信号肽，第二个外显子编码成熟受体蛋白和3'非翻译序列。这两个受体基因还与位于5q13的两个人类基因以及位于小鼠基因组同线区域13D2的两个小鼠基因共有一个基因座。这些技术还表明，这两个基因之间的物理距离小于100 kb。
凝血酶受体和PAR2基因具有相同的结构且在基因组中彼此相距约100 kb，这一事实表明这些基因起源于基因复制事件。这些结果定义了一个新的蛋白酶激活受体基因簇，可能会在其中发现新的家族成员。
