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Regulation of Folate Receptor Raft Recycling
| Title: | Regulation of Folate Receptor Raft Recycling |
| Author: | Elnakat, Hala |
| Description: | FR quantitatively recycles, within minutes, between the cell surface and endocytic compartments via a Cdc42-regulated endocytic pathway. In this study, we investigated the molecular mechanism by which FR recycling is regulated by PMA since its effects on physiological processes are known to stimulate cell signaling through the second messenger diacylglycerol. Phorbol-12-myristate-13-acetate causes a dose dependent increase in the number of receptor molecules residing on the cell surface by inhibiting internalization. Moreover, the release of the intracellular calcium stores which is triggered by the ionophore cyclopiazonic acid results in a doubling of cell surface FR thereby simulating the effect of PMA. Since intracellular calcium is needed for externalization of FR, the PMA effect is probably mediated by a classical PKC isoforms. Western blot of total MA104 cell lysates and quantitative real time RT-PCR show that PKCá is the only classical PKC that can be detected in these cells. Overexpression of wild-type PKCá results in an increase in PMA-induced FR externalization relative to the control cells overexpressing GFP. Knockdown of PKCá using retrovirus or overexpression of a dominant negative PKCá mutant or a constitutively active PKCá which is targeted to the membrane but not specifically to rafts abrogate the PMA effect on FR recycling. These data further suggest that PKCá, targeted to specific membrane microdomains, is one of the key players in mediating the phorbol ester effect on FR recycling in MA104 cells. In order to identify other candidate proteins involved in FR recycling, we purified FR-rich rafts using an immobilized biotinylated folate probe. Among the proteins identified, RACK1 mediates the PMA effect on FR recycling in MA104 cells. The association of RACK1 with FR-rich rafts was further confirmed by electron microscopy in situ in MA104 cells. Though RACK1 has been reported to be present in the cytosol and in caveolae, no evidence for its association with rafts has been documented until now. One of the other proteins found to be associated with FR-rafts is the PKC substrate, annexin II. Unlike RACK1, annexin II is required for the internalization of FR rafts. Finally, the activation of Cdc42 is not diminished by PMA treatment, and therefore is not a likely mediator of the inhibition of raft internalization. These detailed studies provide a comprehensive molecular picture of the effect of PMA on FR recycling in MA104. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=mco1174569209
http://hdl.handle.net/2374.OX/18029 |
| Date: | 2007 |
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