Functional analysis of a novel receptor for lysophosphatidic acid (LPA)

B103大鼠神经母细胞瘤细胞对溶血磷脂酸（LPA）没有反应，用LPA1或LPA4（也称为p2y9/GPR23）表达载体稳定转染。我发现，在LPA的作用下，LPA4介导了B103-LPA4细胞的Ca^<2+>反应。此外，LPA4被证明可以激活Rho小GTPase，导致细胞圆缩和细胞聚集等形态学变化。在LPA刺激下，B103-LPA1细胞虽然也呈圆润状，但其圆润程度明显低于B103-LPA4细胞。在细胞聚集方面，激活LPA1未引起任何形态学改变。我观察到，与LPA1不同，LPA4不与百日咳毒素敏感的Gi/o蛋白偶联，这可能是LPA1和LPA4之间这些功能差异的原因。由于LPA1在大脑形成和神经性疼痛等神经系统中起着至关重要的作用，因此我的数据提出了LPA4在体内也可能具有神经元功能的可能性。制备了兔抗LPA4合成肽的多克隆血清。事实上，在胚胎第12天的小鼠大脑中，通过抗血清Western分析成功检测到富含LPA4 mRNA的LPA4蛋白，并对表达LPA4的B103细胞进行了流式细胞术分析。通过同源重组，研究了海洋胚胎干细胞中LPA4基因的断裂。将3个携带适当同源重组事件的胚胎干细胞克隆（未随机整合靶向载体）注入囊胚。来自三个克隆中的一个的嵌合体导致目标等位基因的种系传播，这被Southern分析证实。

B103 rat neuroblastoma cells, which are unable to respond to lysophosphatidic acid (LPA), were stably transfected, with the expression vector for either LPA1 or LPA4 (also known as p2y9/GPR23). I found that, in response to LPA, LPA4 mediated Ca^<2+> responses in B103-LPA4 cells. Furthermore, LPA4 was shown to activate Rho small GTPase, resulting in the morphological changes including cell rounding and cell aggregation. Although B103-LPA1 cells also became rounded upon LPA stimulation, their degree of cell rounding was significantly less than that of B103-LPA4 cells. As for cell aggregation, LPA1 activation did not induce any morphological changes. My observation that, unlike LPA1, LPA4 did not couple to pertussis toxin-sensitive Gi/o proteins may account for these functional differences between LPA1 and LPA4. Because LPA1 plays critical roles in the nervous system such as brain formation and neuropathic pain, my data raise the possibility that LPA4 also may have neuronal functions in vivo.A rabbit polyclonal antiserum was raised against a synthetic peptide derived from mouse LPA4. Actually, LPA4 protein was successfully detected by the antiserum in Western analysis of mouse brain at embryonic day 12, which was rich in LPA4 mRNA, and flow cytometry analysis of the LPA4-expressing B103 cells.The LPA4 gene was disrupted in marine embryonic stem (ES) cells by homologous recombination. Three clones of ES cells carrying the proper homologous recombination events without random integration of the targeting vector were injected into blastocysts. Chimeras from one of the three clones resulted in germline transmission of the targeted allele, which was confirmed by Southern analysis.