UCLA

Activation of cell surface receptors by ligands leads to the recruitment of a number of signaling molecules that mediate cellular responses. RAS-GTPases are signaling proteins that bring about major changes in cell growth, differentiation, gene expression and cytoskeletal organization upon receptor stimulation. The RAS effector RIN1 is involved in receptor trafficking and signaling. RIN1 is recruited to the receptor following ligand stimulation, but the function of its individual effector domains in this process remained uncharacterized. RIN1 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase RAB5, which is involved in the formation of early endosomes. RIN1 can also directly bind and activate ABL tyrosine kinases, which have been implicated in cellular actin remodeling and receptor trafficking. RIN1 also forms a complex with STAM, an ESCRT complex protein, which favors maturation of receptor-containing endosomes. As all of RIN1's known effectors are involved in modulating receptor trafficking, we resorted to examine the role of individual domains in receptor internalization and fate. Chapters two and three debrief the mechanisms by which RIN1 effectors function in an integrated manner to regulate receptor trafficking and corresponding cellular responses.

Using domain-specific mutants of RIN1, we show that the RAB5 and ABL signaling pathways mediate opposing functions, maintaining a balance in the route and rate of receptor internalization. RAB5 promotes actin remodeling, EGF induced macropinocytosis and migration, facilitating receptor degradation. ABL kinases, on the other hand, prevent receptor degradation and migration towards EGF. Our study also shows that regulation of RIN1 localization by the 14-3-3 proteins is a major factor in determining signaling intensity. We characterized a novel binding partner of RIN1, namely, BIN1, a BAR domain protein involved in membrane bending.

Intracellular pathogens invade host cells by exploiting host cell surface receptors and signaling pathways. Listeria monocytogenes in a food-borne pathogen that binds host MET to invade epithelial cells and modulates RAB5 GTPases at several stages of the infection. The effect of host RAB5 regulators on pathogenesis remained uncharacterized. We analyzed the role of RIN1, a major RAB5-GEF in epithelial cells, in the process of bacterial invasion and spread (chapter four). This study leads the way to determining the mechanism of pathogenesis of several pathogens that depend on host RAB5-GTPases for effective infection progress. Finally, chapter five discusses the significance of our studies from a therapeutic viewpoint in the context of cancers and infectious diseases.