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

Abstract

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.

An early endosome–derived retrograde trafficking pathway promotes secretory granule maturation

“…4 , D′, E′, and F′), raising the question of how SGs acquired CD63-GFP if the retrograde trafficking pathway was compromised. We hypothesized that immature SGs would acquire some CD63-GFP as they bud from the TGN, because CD63 is normally secreted to the plasma membrane before entering the endosomal system ( Duffield et al, 2003 ; Pols and Klumperman, 2009 ; Schulze et al, 2017 ; Yu et al, 2016 ). This is likely the case, as we observed CD63-GFP on immature SG membranes in early L3 salivary gland cells from both WT controls and PI4KIIΔ mutants ( Fig.…”

Tetraspanin Assemblies in Virus Infection

“…Many viruses, including HIV, IAV, HPV, and LUJV, localize to CD63-positive endosomes during entry ( 35 , 46 , 55 , 81 , 84 ). CD63 is most abundant in late endosomes or multivesicular bodies (MVBs) ( 85 ) and involved in the membrane organization and trafficking of cellular transmembrane proteins that interact with viruses such as HIV-receptor component CXCR4 ( 83 , 86 , 87 ). Therefore, a functional involvement of CD63 in viral fusion and transport is conceivable.…”

Organic Cation Transporters in Human Physiology, Pharmacology, and Toxicology

“…Through a mating-based split-ubiquitin system screening, it has been found that tetraspanin CD63, a four transmembrane domains protein that facilitates cell adhesion and motility, was a protein partner of OCT1, -2 and -3 [ 47 , 48 ]. It has also been demonstrated that CD63 was critical for the correct basolateral localization of OCT2 in proximal tubular cells [ 47 ]. The motif sequence that might be involved in the basolateral sorting of OCT1, -2 and -3 is not known.…”