| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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.
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