hOCT2 is a transmembrane protein with high renal expression involved in the polyspecific transport of endogenous and exogenous organic cations (monoamine substrates und drugs, respectively) across the cell membrane. In the kidney, hOCT2 is expressed on the basolateral membrane of proximal tubule cells, where it mediates the first step in the excretion of organic cations. Since the function of hOCT2 is regulated by several different signalling pathways (Biermann et al., Am. J. Physiol. Cell Physiol, 290: 1521–31, 2006), the identification of proteins that directly interact with hOCT2 is important to elucidate the mechanisms of hOCT2 function and regulation. To identify interaction partners of hOCT2, we established a mating based split ubiquitin Yeast-Two-Hybrid (YTH) system technique, which, in contrast to classical YTH systems, allows to detect interactions taking place directly at the cell membranes. Using this system, we could identify the lysosomal associated protein transmembrane 4 alpha (LAPTM4A) as interaction partner of hOCT2. LAPTM4A is a transmembrane protein that is associated with lysosomes and late endosomes. This protein mediates the transport of several substances that are also substrates of organic cation transporters (organic cations, nucleosides) and is supposed to regulate the compartmentalization of amphiphilic solutes in the cell. LAPTM4A has been demonstrated to alter cell sensibility towards drugs (e.g. ethidium bromide, estradiol). We confirmed hOCT2/LAPTM4A interaction with a pulldown assay based on binding of His-tagged LAPTM4A to talon sepharose beads, incubation of the beads-LAPTM4A complex with lysates from HEK cells stably expressing hOCT2 and hOCT2/LAPTM4A detection by Western blot analysis in eluates. The interaction of LAPTM4A with hOCT2 has also a functional meaning because transfection of LAPTM4A in HEK-cells stably expressing hOCT2 decreased significantly the uptake of the fluorescent organic cation 4–4-dimethylamino-styryl-N-methylpyridinium (ASP⁺) in comparison to cells transfected only with hOCT2 (-233% n=31). Transfection with an empty vector showed no effect on ASP⁺-uptake (+44% n=24 compared to control cells). These results suggest a negative effect of LAPTM4A on hOCT2. This interaction could have an important regulatory meaning for hOCT2 activity both in vitro and in vivo, because using RT-PCR, we showed that LAPTM4A is expressed in human proximal tubules, where hOCT2 is also localized. Further investigations are necessary to elucidate the functional mechanisms and the specificity of this interaction (effect of LAPTM4A on the trafficking of hOCT2 or on the lysosomal sequestration of hOCT2 substrates).

Supported by the Deutsche Forschung Gemeinschaft (CI 107/4–1)