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UNDERSTANDING AND MANIPULATING ENDOCYTOSIS-DEPENDENT SIGNALING CIRCUITS
Signal transductions are essential processes for living cells to react to environmental stimuli adequately, and they need to be tightly regulated as they can affect cell survival and cell fate determination. Since many of these signaling events rely on the presence of receptors on the cell membrane, members of endocytic proteins play critical regulatory roles in signaling via changing the localization of the receptors. In particular, endocytic adaptors are the linkers that connect membrane cargo and other members of endocytic machinery to accomplish the process. We focused on the roles of the endocytic adaptors epsins and their cargoes in signaling, as both epsins’ transmembrane and cytosolic cargoes participate in signaling pathways.
We investigated the molecular mechanism of how epsins recognize specific ubiquitinated membrane cargoes among other ubiquitinated membrane proteins. Through genetic, biochemical, and cell biological approaches, we identified the first yeast transmembrane cargo, Ena1, a P-type ATPase sodium pump. We report that the simultaneous presence of phosphorylation and ubiquitination on the Ena1 are required for epsin-specific recognition. We also demonstrated that post-translational modifications are Yck1/2 and Art3-Rsp5 dependent, and the spatial arrangement of the modifications is essential.
In addition to the regulation of signaling pathways through internalizing transmembrane cargoes, epsins are also involved in the regulation of Rho GTPase signaling pathways. Through direct interaction, epsins inhibit activities of their cytosolic cargoes, Rho GTPase activating proteins (RhoGAPs). Ocrl1 is one of the epsin interacting RhoGAP domain-containing proteins. The deficiency of Ocrl1 leads to a lethal developmental disease called Lowe syndrome (LS). While the patients display developmental problems affecting the brain and eyes, they also suffer from kidney dysfunction that results in death. The pathological mechanism is currently obscure and no cure, partly due to the lack of an adequate cell model from the affected tissues. We generated the first iPSC model from fibroblasts of LS patients and normal individuals and further generated kidney cells from these iPSCs. Consistent with observations obtained from LS fibroblasts, the LS iPSC derived kidney cells from patient cells also have a deficiency in ciliogenesis.Further, we discovered that Six2, a crucial transcriptional factor in kidney development, is mislocalized to the Golgi-apparatus in patient iPSC-derived kidney cells as well as in an OCRL1 K.O. proximal tubular cell line. Disproportional cell lineage differentiation is also observed in the patient group. The iPSC model provides an opportunity to investigate the differences between normal and disease cell differentiation in all the affected tissues, generate organoids, and develop cell replacement therapies.