Identification of the Tetraspanin CD9 as an Interaction Partner of Organic Cation Transporters 1 and 2.

Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 (SLC22A1/hOCT1) and hOCT2 (SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63 and CD9. Moreover, we confirmed interaction of hOCT2 with CD9 by fluorescence-activated cell sorting coupled with Förster resonance energy transfer analysis. Together with other proteins, tetraspanins build "tetraspanins webs" in the plasma membrane, which are able to regulate cellular trafficking and compartmentalization of interacting partners. While CD63 was demonstrated to mediate the localization of the hOCT2 to the endosomal system, we show here that co-expression of hOCT2 and CD9 led to strong cell surface localization of the transporter. These data suggest that tetraspanins regulate the cellular localization and function of OCTs. Co-localization of CD9 and hOCT was confirmed in tissues endogenously expressing proteins, highlighting the potential biological relevance of this interaction.

Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.

CD63 is a ubiquitously expressed member of the tetraspanin superfamily. Using a mating-based split-ubiquitin-yeast 2-hybrid system, pull-down experiments, total internal reflection fluorescence microscopy, Förster resonance energy transfer, and biotinylation assays, we found that CD63 interacts with human organic cation transporter 2 (hOCT2), which transports endogenous and exogenous substrates, such as neurotransmitters and drugs in several epithelial cells. CD63 overexpression affects cellular localization of hOCT2 expressed in human embryonic kidney (HEK)293 cells. Studies with CD63-knockout mice indicate that in renal proximal tubules, CD63 determines the insertion of the mouse ortholog of the transporter into the proper membrane domain and mediates transporter regulation by trafficking processes. In polarized Madin-Darby kidney canine kidney (MDCK) epithelial cells, CD63 and hOCT2 colocalize with the small GTPase Rab4, which controls the rapid recycling from sorting endosomes back to the cell surface. Suitable negative and positive control experiments were performed for each experimental approach. Empty vector transfected cells and wild-type mice were used as control. CD63 seems to play a role in the recycling of hOCT2 from endosomes to the basolateral membrane in polarized epithelia. These data indicate that CD63 has a previously uncharacterized function in regulating trafficking of specific membrane proteins in polarized cells.-Schulze, U., Brast, S., Grabner, A., Albiker, C., Snieder, B., Holle, S., Schlatter, E., Schröter, R., Pavenstädt, H., Herrmann, E., Lambert, C., Spoden, G. A., Florin, L., Saftig, P., Ciarimboli, G. Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.