The signaling pathways emanating from TNF receptor (TNFR1) are well studied. However, little is known about the intracellular trafficking pathway of this important cytokine receptor. Preliminary data show that treatment of kidney epithelial cells and other cell types with the cytokine TNF stimulates the translocation of TNFR to the cell surface. This ligand-stimulated translocation can be attenuated by blocking myosin II motor activity, suggesting a role for this molecular motor in regulating the plasma membrane levels of TNFRI. In this application, three aims are proposed to test the hypothesis that specific forms of MLCK and myosin heavy and regulatory fight chains, regulate the trafficking of TNFR1 from the Golgi to the plasma membrane and to define the intracellular pathway regulated by myosin II motor activities.
These specific aims are 1) to determine if a specific form of MLCK is involved in regulating TNF-induced translocation of TNFR1 in kidney epithelial cells. The role of a newly characterized 100 kDa MLCK and a 220 kDa MLCK will be examined in trafficking TNFR1 to the plasma membrane by expression of dominant negative forms of these kinases and by antisense depletion.
Aim 2) will determine the association between a previously identified and a new myosin regulatory light chain (RLC) and myosin heavy chains IIA and lIB. Expression studies using dominant negative and phosphomimetic RLCs will determine the relationship between the RLCs and trafficking of TNFR1 to the plasma membrane.
Aim 3) will define the interactions between MHC-IIA, TNFR1 and Rab8 and to determine their roles in TNF-induced trafficking of TNFR1. These studies will provide fundamental new information about the role of the acto-myosin cytoskeleton in vesicular trafficking that will be important for developing therapeutic strategies for renal failure.
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