Date of Award

11-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Xin Zhang, M.D.

Committee

John V. Cox, Ph.D. Eldon E. Geisert, Ph.D. Lisa K. Jennings, Ph.D. Andrzej T. Slominski

Keywords

CD82, internalization, intracellular trafficking, tetraspanin, YXXΦ motif

Abstract

CD82, a tetraspanin, is a tumor metastasis suppressor. Tetraspanins are membrane spanning proteins that play critical roles in diverse biological and pathological processes, e.g., the regulation of cancer metastasis. CD82 is ubiquitously expressed in various types of tissues, but its expression becomes down-regulated or lost in a majority of metastatic tumors. It inhibits tumor metastasis without affecting primary tumor growth. Cancer patients whose tumors contain CD82 exhibit minimal metastasis. We know that CD82 functions as a tumor metastasis suppressor but the mechanism by which this occurs is largely unknown. CD82 can be found on the plasma membrane as well as in intracellular compartments, and CD82 undergoes endocytosis. The intracellular trafficking of proteins plays a key role in their functions. The mechanism by which CD82 is able to traffic remains not well known. Notably, CD82 contains an YXXΦ internalization and intracellular trafficking motif in its C-terminal cytoplasmic domain.

Previous reports suggest that the mutation of the YXXΦ motif of a tetraspanin will disrupt internalization and subsequent trafficking. We have established the stable transfectant of CD82 YVAA mutant, which contains a mutation in the YXXΦ motif of CD82.

This research focuses on the roles of CD82’s YXXΦ motif 1) in intracellular trafficking of CD82 and its associated proteins as well as 2) in the CD82-mediated inhibition of the migration of metastatic tumor cells. Results from the internalization assays reveal that the YXXΦ motif is not required for the internalization of CD82, nor does the mutation of this motif have drastic effect on the steady state distribution of CD82. Results from the intracellular trafficking and recycling assays reveal that there is a defect in the intracellular trafficking of CD82 upon the mutation of its YXXФ motif. We found that both CD82 WT and CD82 YVAA mutant trafficked to the late endosomes/lysosomal compartments. We also observed that the recycling of CD82 proteins is Rab 11-mediated process and that the mutation of the YXXΦ motif significantly decreases the CD82 YVAA release via exosomes. Our study also revealed that the CD82 YVAA mutant traffics to and accumulates within the lysosomal compartments within the cells. Regarding the role of CD82 trafficking in cell movement, we found that that the mutation of CD82 YXXΦ motif results in a loss of function in CD82-mediated inhibition of collective cell migration and cell invasion but appears not to affect CD82-mediated inhibition of solitary cell migration. In addition, CD82 YVAA mutation increases cell-cell adhesion in a calcium dependent manner, which likely results from altered trafficking of cell-cell adhesion proteins.

From the results obtained in this study, we conclude that the YXXΦ motif of CD82 is not essential for CD82 internalization but rather regulates CD82 trafficking to the late endosome where CD82 is recycled back to the plasma membrane or secreted into the extracellular environment via exosomes. CD82 trafficking also regulates the trafficking and function of cell adhesion proteins. Furthermore, the proper trafficking and recycling of CD82 plays a key role in maintaining its motility suppressive function. Further studies will determine 1) the molecular mechanism by which CD82 trafficking regulates the behavior of cell adhesion proteins and 2) the effect of aberrant CD82 trafficking on tumor metastasis in animal models.

DOI

10.21007/etd.cghs.2012.0259

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