Regulation of membrane trafficking in polarized epithelial cells Date Added: 4/29/2009 11:49:00 AM
Last Updated: 5/1/2009 2:41:00 PM
Description of projects available to graduate students:
The laboratory’s primary research interest is the molecular basis for the polarized expression and trafficking of proteins in epithelial cells. We are presently utilizing three apical membrane proteins (mucin-like MUC1, the epithelial sodium channel named ENaC, and the small hydrolase gamma-glutamyltranspeptidase) as models to identify the signal(s) that are used by the cell to deliver, maintain and recycle them at that surface. Rotation students could be involved in studies in their project of interest.
Human MUC1 functions to modulate the membrane trafficking and signaling of the EGF receptor. MUC1 exhibits predictable anti-adhesive properties normally attributed to long extended mucin-like molecules with extensive glycosylation, and adhesive properties likely based on specific O-glycan structures. Our previous published studies of MUC1 indicate that it is internalized by clathrin-mediated endocytosis that requires binding of adaptors AP-2 and Grb2, while recycling is dependent on Cys-palmitoylation. Its glycosylation directly impacts its apical targeting, endocytosis and stability. Present studies are focused on the role of galectins in regulation of MUC1 membrane trafficking and stability.
The epithelial Na+ channel (ENaC) is the rate limiting step for transepithelial Na+ transport across high resistance, Na+ transporting epithelia of the renal distal tubule and airway. Our previous publications describe the novel role of proteolysis in activating the channel by release of short inhibitory peptides. Our current studies are focused on the role of Cys-palmitoylation in regulating ENaC membrane trafficking, especially its exit from the endoplasmic reticulum.
Gamma-Glutamyltranspeptidase (GGT) functions in the hydrolysis of extracellular glutathione and represents one of the many microvillar hydrolases localized on the apical surface of polarized epithelial cells. The apical targeting signal for this class of single-pass transmembrane proteins is unknown. For other apical proteins, N-glycans, O-glycans or specific residues within the transmembrane domain are required for proper targeting. Thus, our present studies utilize the expression of recombinant mutated forms of the rat GGT in polarized cell lines (MDCK cells) to characterize the apical targeting signal of this protein. Characterization of the disulfide bridges in GGT has also led to studies of the mechanism of GGT autocatalytic activation within the endoplasmic reticulum and its impact on the redox balance within this unique compartment.
Techniques graduate student will learn:
The student would utilize cultured MDCK cells, CHO cells or tumor cell lines to characterize the expression of wild-type or mutated forms of ENaC, MUC1 or GGT utilizing standard techniques of such as immunofluorescence microscopy, metabolic radiolabelling, protein immunoprecipitation, SDS-PAGE and Western blotting.
Molecular Genetics and Developmental Biology
Cell Biology And Molecular Physiology
Return to list