This project is oriented towards determining factors that regulate the fate of transmembrane proteins both on the cell surface and in the secretory pathway. There is evidence from yeast that a ubiquitin protein ligase (Rsp5) plays a significant role in the internalization and subsequent degradation in vacuoles of several multi-membrane spanning transporters and receptors. We have recently cloned one murine homolog of Rsp5 and more recently obtained cDNAs encoding another. Studies are underway to determine whether overexpression of either active or inactive forms of these enzymes affects the cell surface expression of the T cell antigen receptor and tyrosine kinase-encoding growth factor receptors. Additional studies are aimed towards evaluating the role of c-Cbl as a ubiquitin protein ligase in the ubiquitination and internalization of receptors, particularly the T cell antigen receptor and the epidermal growth factor receptor. Other aspects of this project are oriented toward determining factors that influence the fate of proteins along the secretory pathway. We have now established that degradation of two subunits of the T cell antigen receptor from the ER involves ubiquitination and targeting to proteasomes. Inhibition of proteasome function results in the accumulation of these subunits in membrane-bound forms. Interestingly, the fate of these proteins is also affected by their glycosylation state. GFP-chimeras of TCR subunits have now been generated to allow for analysis of their fates in real time. In yeast, degradation from the ER has been shown to involve two enzymes of the ubiquitin conjugating system known as UBC6 and UBC7. UBC6 is a type-IV membrane anchored protein while UBC7 has no membrane anchor. We have now isolated mammalian homologs of both UBC6 and UBC7 and are evaluating their subcellular localization, physical and functional interactions, and role in targeting of T cell antigen receptor subunits for degradation from the ER.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
United States
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