BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 90–94 (1997) ARTICLE NO. RC976588 Identification of a Unique Monocarboxylate Transporter (MCT3) in Retinal Pigment Epithelium Heeyong Yoon,* Albertina Fanelli,† Evelyn F. Grollman,† and Nancy J. Philp* ,1 *Pennsylvania College of Optometry, Philadelphia, Pennsylvania 19141-3399; and †Laboratory of Biochemistry and Metabolism, NIDDK, NIH, Bethesda, Maryland Received March 28, 1997 interphotoreceptor space (IPS), the RPE will influence The retinal pigment epithelium transports lactate photoreceptor metabolism and viability (1,4,7). between two tissue compartments, the interphotore- Recently, we identified an integral membrane pro- ceptor matrix and the choriocapillaris. In this report tein in the basolateral membrane of differentiated we describe a 2.45-kb cDNA isolated from a chick cDNA chick RPE cells (11). The protein was identified using RPE library that encodes a membrane protein found a monoclonal antibody (MAb 3C4) and called REMP only in RPE cells. The deduced protein has 542 amino (retinal epithelial membrane protein). REMP was acids with twelve putative membrane spanning do- found only in chick RPE cells and not in other chick mains. The cDNA has been designated MCT3 based on tissues such as neural retina, intestine, kidney or liver its 45% identity in amino acid sequence and structural (11). In this report, we describe the heterologous ex- similarity with the monocarboxylate transporters pression of a full length cDNA clone that encodes this MCT1 and MCT2. Stable transfectants (pCl-neo/MCT3), protein. The deduced amino-acid sequence of the cDNA made in a rat thyroid epithelial cell line (FRTL-5), ex- (isolated from a chick RPE expression library) is homol- press MCT3 RNA. Transfectants had enhanced pyr- ogous with the recently cloned monocarboxylate trans- uvate uptake (used as a measure of lactate uptake) porters MCT1 and MCT2 (12,13). The heterologous ex- which was proton-dependent and inhibited by a-cy- ano-4-hydroxycinnamate. In summary, MCT3’s unique pression of a cDNA in FRTL-5 cells demonstrated that expression in RPE cells, multiple potential phosphory- REMP is a proton coupled monocarboxylate trans- lation sites, and basolateral distribution suggest that porter and is designated MCT3. MCT3 may regulate lactate levels in the interphotore- ceptor space.  1997 Academic Press MATERIALS AND METHODS Isolation of MCT3 clones. Preparation of the chick RPE cDNA library used in this study is described elsewhere (11). The library Retinal pigment epithelium (RPE) forms the outer was screened with a digoxigenin-labeled random primed DNA probe blood-retinal barrier and regulates the vectorial move- prepared from the 300 bp EcoRI-DraII fragment of REMP cDNA (11). Positive clones were plaque purified; phage DNA was digested ment of fluid, ions, and metabolites between the neural with EcoRI; and cDNA inserts subcloned into pBluescript SK- (Stra- retina and the choroidal circulation (reviewed in 1). tagene). DNA was sequenced using ABI 373A Stretch sequencer at A close association between RPE and neural retina is the Genetics Core Facility at the University of Pennsylvania, Phila- established early in development and is essential for delphia, PA. The sequence of one clone, with a 2.2 kb insert DNA, maintaining photoreceptor cell viability (2,3). The added 710 nucleotides to the 5 proximal end of the previously re- ported REMP sequence (11). The combined cDNA sequence was maintenance of visual cell function depends on glycoly- named MCT3 based on its sequence homology to MCT1 and MCT2. sis. The neural retina with its high rate of metabolism, Sequence comparisons were generated using the Genetics Computer uses glucose and produces substantial quantities of lac- Group (Madison, WI). tate, in both light and dark (4-6). Since the outer retina Stable transfection of FRTL-5 cells. Characteristics and propaga- is avascular, the RPE moves glucose and lactate into tion of FRTL-5 cells (ATCC CRL 8305, F1 clone), are described else- and out of the subretinal space for use by the retina where (14,15). To generate stable transfectants, MCT3 cDNA was (7-10). By regulating the lactate concentration in the ligated into the EcoRI site of the mammalian expression vector, pCI- neo (Promega). FRTL-5 cells were trypsinized and suspended in 0.35 ml of cytomix (16) at a concentration of 4.5 1 10 6 cells per cuvette with 10 mg of plasmid DNA. The cells were electroporated at 300 1 To whom correspondence should be addressed at Pennsylvania College of Optometry, 1200 W. Godfrey Ave., Philadelphia, PA 19141- Volts, 960 mF and plated in one 100 mm dish in medium containing 0.5% penicillin/streptomycin (Biofluids Inc.). Selection with 400 mg/ 3399. Fax: 215-276-6081. E-mail: nphilp@hslc.org. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 90 AID BBRC 6588 / 692a$$$401 04-15-97 14:24:32 bbrcg AP: BBRC