The regulated recycling of endocytosed membrane proteins at the plasma membrane is an important intracellular trafficking process that can regulate the copy number of proteins at the membrane and thereby modulate their function and the physiology of the tissue as a whole. The Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes rapid and efficient recycling at the apical plasma membrane in polarized epithelial cells. The cellular mechanisms that facilitate CFTR recycling are understood poorly, especially in polarized cell systems, yet this process ensures the proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Using a physiologically relevant model that recapitulates the in vivo secretory functions of the intestinal epithelia, I tested the hypothesis that distinct members of the Rab family of GTPases mediate the recycling of CFTR at the apical plasma membrane and that this process facilitates transepithelial anion secretion by maintaining a physiologically functional surface density of CFTR channels. The role of the Rab11 isoforms in CFTR recycling was investigated using the colonic epithelial cell line, T84, and rat intestine for in vivo studies. Immuno-isolation of Rab11 vesicles revealed endogenous CFTR within both the Rab11a and Rab11b compartments; however, only perturbing Rab11b expression or function attenuated the CFTR-mediated, cAMP-activated anion efflux. In polarized epithelial monolayers, mutant Rab11b inhibited the forskolin-stimulated transepithelial anion secretion by reducing the apical membrane density of CFTR. Biotin protection assays revealed a dependence of CFTR recycling on functional Rab11b, not Rab11a, demonstrating the selective requirement for the Rab11b isoform. Therefore, apical CFTR copy number is regulated by Rab11b-mediated recycling, which facilitates the agonist-stimulated, transepithelial anion response in polarized intestinal epithelial cells. The identification of Rab11b as a mediator of recycling reveals an apical recycling itinerary unidentified previously and emphasizes the importance of elucidating the membrane recycling itineraries in polarized epithelial cell models, which mimic the normal physiology of tissue. Understanding these processes within relevant models have direct implications for understanding normal cell biology and tissue physiology and form a foundation for future clinical therapies designed to correct disease processes arising from improper membrane recycling.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-11232009-174209 |
| Date | 18 December 2009 |
| Creators | Silvis, Mark Robert |
| Contributors | Ray A. Frizzell, Ph.D., John P. Johnson, M.D., Gerard L. Apodaca, Ph.D., Daniel C. Devor, Ph.D., Neil A. Bradbury, Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-11232009-174209/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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