Lysophosphatidic acid (LPA) is a mitogenic lipid that enhances cell growth, proliferation and motility through binding and activation of at least four receptors, LPA1/Edg2, LPA2/Edg4, LPA3/Edg7, and PPAR and #947;. Here, we show that LPA stimulation inhibits the cell cycle regulator and tumor suppressor, p53. Ten M LPA reduced the cellular levels of total p53 and p53 phosphorylated at serine 15 by approximately 50% in A549 cells and this effect was sustained for at least 6 h. This resulted in a corresponding decrease in p53-mediated transcription. Transient-transfection of the Edg-family LPA receptors, LPA1-3 in HepG2 cells, which do not respond to LPA, also showed this inhibitory response. The response was specific to LPA receptors since neither Gi-coupled M2 muscarinic acetylcholine receptors, nor a mutant LPA1 receptor (LPA1 R124A), which is unable to bind LPA, inhibited p53 activity. Both transient-transfection of the LPA-degrading lipid phosphate phosphatase-1 (LPP-1), or exogenous addition of phospholipase B, which decreases exogenous lysophosphatidate, reversed the LPA receptor-induced decrease in p53-mediated transcription. Although pertussis toxin did not prevent the inhibition of p53, a mutant LPA1 receptor (LPA1 and #8710;361), which lacks the C-terminal PDZ-binding domain, failed to inhibit p53 function. This establishes LPA-mediated inhibition of p53 function requires an interaction with PDZ-containing proteins. These data establish a novel role for LPA-mediated receptor activation in diminishing p53 activity; which, in addition to LPAs well-characterized effects on growth-promoting signaling pathways, is likely to contribute to the survival and proliferation of cancer cells.
Of the Edg-family LPA receptors, the LPA1 receptor is the most widely expressed. In the next study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. LPA treatment induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removing agonist following incubation resulted in LPA1 recycling back to the surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). Finally, our results indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/6869 |
| Date | 26 April 2005 |
| Creators | Murph, Mandi Michelle |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | 10292219 bytes, application/pdf |
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