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

Abstract

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.

Expression and Function of Organic Cation Transporter 2 in Pancreas

“…Transfection of GLUT2 decreases the transport activity of hOCT2 overexpressed in HEK cells, suggesting that the hOCT2-GLUT2 interaction detected using the mbSUS (Snieder et al, 2019) may have functional consequences. However, in pull-down experiments using GFP-trap and HEK cells transfected with hOCT2-GFP [the GFP tag is localized at the carboxy-terminus of the transporter and does not change its function, as already demonstrated in Brast et al (2012)] it was not possible to confirm a direct hOCT2-GLUT2 interaction.…”

“…However, in enterocytes after a sugar meal (Leturque et al, 2009) and in renal proximal tubules in dependence from increasing glucose concentrations (Marks et al, 2003), GLUT2 can translocate to the apical membrane. Using a mating-based split-ubiquitin-system (mbSUS), a protein-protein interaction between GLUT2 and the human OCT2 (hOCT2) was observed (Snieder et al, 2019). Because hOCT2 is a transporter for metformin (Song et al, 2008;Konig et al, 2011) and metformin also acts on pancreas (Yang et al, 2017), in this brief research report we investigated, whether OCT2 is expressed in pancreatic tissue and whether GLUT2 and glucose can regulate its activity.…”

Regulation Mechanisms of Expression and Function of Organic Cation Transporter 1

“…A screening performed using the mating-based split-ubiquitin system, a special yeast-two-hybrid technique, able to detect protein-protein interactions taking place in the plasma membrane, identified 24 potential interaction partners for hOCT1 (Snieder et al, 2019). According to gene ontology annotations, the interacting proteins are associated mainly with transport processes, vesicle-mediated transport, signaling pathways, protein modification, homeostatic processes, and cell adhesion (Snieder et al, 2019).…”

“…A screening performed using the mating-based split-ubiquitin system, a special yeast-two-hybrid technique, able to detect protein-protein interactions taking place in the plasma membrane, identified 24 potential interaction partners for hOCT1 (Snieder et al, 2019). According to gene ontology annotations, the interacting proteins are associated mainly with transport processes, vesicle-mediated transport, signaling pathways, protein modification, homeostatic processes, and cell adhesion (Snieder et al, 2019). The cellular distribution of the identified interaction partners may reflect hOCT1 cellular processing: they are localized in the plasma membrane (CD9 (tetraspanin-29), CYSTM1 (cysteine-rich and transmembrane domain containing protein 1), and PDZKP1), in the endoplasmic reticulum (KRTCAP2 (keratinocyte-associated protein 2), SERP1 (stress-associated endoplasmic reticulum protein 1), VAMPB (vesicle-associated membrane proteinassociated protein B isoform 1), and TMEM147 (transmembrane protein 147), in the Golgi system (CHST12 (carbohydrate (chondroitin 4) sulfotransferase 12) and TMBIM4 (transmembrane protein 41B), in endosomes and lysosomes (CD63 (tetraspanin-30) and LAPTM4A (lysosomal associated protein transmembrane 4 α), and in mitochondria (FIS1 (fission, mitochondrial 1 protein), GHITM (growth hormone-inducible transmembrane protein), and SLC25A11 (solute carrier family 25, member 11)).…”

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway

“…Human CD9 was amplified from pExpress-1-CD9, CD9 (clone IMAGp998A1815788Q, imaGenes, Berlin, Germany) by PCR and subcloned into the XhoI-KpnI site of the pEYFP-C1 (Clontech Laboratories, Mountain View, CA, USA) vector as described before [37] and into the XhoI-KpnI site of the pCMV-HA (Clontech) and pcDNA3.1/ Hygro(−) (Thermo Fisher Scientific) vectors. The ADAM17 wild type (WT) plasmid was kindly provided by Dr. Gillian Murphy (Cambridge, UK) and was described previously [38].…”