Project Details
Projekt
Breakdown of the proteome of organic cation transporters: Implications for function and regulation
Subject Area
Nephrology
Anatomy and Physiology
Anatomy and Physiology
Term
from 2008 to 2016
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 77329949
Polyspecific organic cation transporters (OCT) with their subtypes OCT1, 2, and 3 play an important role in tissue distribution of endogenous and exogenous organic cations and in their renal (in humans mainly mediated by the subtype 2, hOCT2) and hepatic (in humans mainly mediated by the subtype 1, hOCT1) secretion. In polarized tissues OCT are principally expressed on the basolateral plasma membrane. We could show using several independent in vivo and in vitro methods (mbSUS, pull-down, FRET, siRNA, TIRF microscopy and uptake experiments) that membrane expression and function of OCT are regulated by direct interaction with the tetraspanin CD63. We hypothesize that this interaction has different functional implications when taking place in polarized or in non-polarized cells. In non polarized HEK293 cells stably transfected with hOCT2, CD63 seems to recruit the hOCT2 from the plasma membrane to an endosomal pathway. Conversely, in renal proximal tubules (which are made up by polarized cells) from CD63 knockout (CD63 KO) mice, the specific basolateral localization of OCT appears to be lost, being the transporter distributed in small cytosolic vesicles spread across the cells. Moreover, acute regulation of OCT activity by trafficking processes is suppressed in proximal tubules of these animals. For these reasons we conclude that in polarized cells CD63 seems to direct the insertion of the OCT into the proper membrane domain. To test our hypothesis, in this project we will study the relationship between hOCT2 and CD63 in polarized cells of renal origin such as the Immortalized Human Kidney Epithelial (IHKE-1) and/or Madin-Darby Canine Kidney (MDCK) cells stably expressing these proteins. Moreover, to appreciate the interaction dynamics, we will try to establish these cell lines also with inducible protein expression. The role of CD63 in determining plasma membrane expression will be also studied in primary cell cultures from proximal tubules of WT and CD63 KO mice.
DFG Programme
Research Grants
