Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Research Advisor

Anjaparavanda Naren, Ph.D.

Committee

Suleiman W. Bahouth, Ph.D. Larry Pfeffer, Ph.D. Radhakrishna Rao, Ph.D. Donald B. Thomason, Ph.D.

Keywords

CFTR, cystic fibrosis transmembrane conductance regulator, Hsp90, PKA, Mass Spectrometry, Xenopus, Calu-3, HEK-293, Radicicol, Ussing Chamber

Abstract

The hypothesis of this project is that a functionally relevant association exists between CFTR and Hsp90. In testing this hypothesis, (a) the association of CFTR and Hsp90 is confirmed via crosslinking and immunoprecipitation of CFTR from stably transfected HEK-293 cells, electrophoretic separation of cross-linked proteins, and protein identification via SDS-PAGE, western blotting, and mass spectrometry. Also, this hypothesis is tested by (b) functionally assessing the effect of Hsp90 on CFTR function via Ussing chamber analysis, two-electrode voltage clamp analysis, preliminary in vivoanimal studies, and protein phosphorylation. The HEK-293 Flag-CFTR cells used in this study allows for a high degree of protein purification in quantities sufficient for mass spectroscopy, which has not been possible in other cell lines. An approximately 170 kDa band could be detected with CFTR specific antibodies. The HEK-293 Flag-CFTR cells also show increased protein expression levels above that observed in BHK cells transfected with wild-type (WT)-CFTR and CFTRhis10. The Flag-CFTR expression was not only confirmed via immunofluorescent detection, but was also shown to be functional using a standard iodide efflux assay. The HEK-293 Flag-CFTR cells responded to agonists known to activate CFTR (forskolin, IBMX, cpt.cAMP, isoproterenol, and adenosine). Furthermore, these cells express sufficient quantities of protein to allow for mass spectrometric analysis, which has proven difficult in other cell lines. Thus, this cell line is a valuable tool for the biochemical and functional characterization of CFTR and should prove useful for future studies assessing protein-protein interactions as they relate to CFTR. Using cross-linking reagents (DSP & DPDPB), Hsp-90 is shown to be in association with CFTR in stably transfected HEK-293 cells. Hsp90 associates with CFTR under these conditions more prominently than does Hsp70. The amount of Hsp90 in the macromolecular complex with CFTR is sufficient for mass spectrometry identification. An in vitro assay for CFTR transport function (Ussing Chamber) shows that inhibition of Hsp90 with RD, an Hsp90 inhibitor with nanomolar affinity, decreases CFTR mediated Cl- transport. Preliminary in vivo airway fluid clearance studies in mice also show that RD inhibits fluid transport, known to require CFTR. Also, Hsp90 protein increases the two-electrode voltage clamp current measured from Xenopus oocytes expressing CFTR chloride channels. When assessing the phosphorylation status of CFTR, it is seen that under conditions that increase the association of Hsp90, there is also observed an increase in the phosphorylation of CFTR. Conversely, in the presence of increasing concentrations of RD, both the Hsp90 association CFTR and the phosphorylation of CFTR tend to decrease. Taken together these data suggest that Hsp90 can be positively identified in complex with CFTR and can functionally regulate its activity.

DOI

10.21007/etd.cghs.2007.0202

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