The mechanism of carrier-mediated transport of folates in BeWo cells: the involvement of heme carrier protein 1 in placental folate transport

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FRalpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCP1) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FRalpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Ultrastructural localization of adhesion molecules in the healthy

The functional expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and MAdCAM-1 in the choroid plexus is indicative of a role of this structure in the communication of the immune system with the central nervous system (CNS). In order to gain further insight into the possible functions of adhesion molecules expressed in the choroid plexus, we investigated the exact ultrastructural localization of VCAM-1, ICAM-1 and MAdCAM-1 on semithin and ultrathin cryosections of the choroid plexus of healthy mice and of mice suffering from experimental autoimmune encephalomyelitis (EAE). In the healthy choroid plexus VCAM-1 and ICAM-1, but not MAdCAM-1, could be detected on the apical surface of the choroid plexus epithelial cells. During EAE, immunoreactivity for VCAM-1 and ICAM-1 was dramatically increased. Additionally, apical expression of MAdCAM-1 was observed on individual choroid plexus epithelial cells during EAE. At the same time, VCAM-1, ICAM-1 or MAdCAM-1 were never present on the endothelial cells of the fenestrated capillaries within the choroid plexus. The polar expression of VCAM-1, ICAM-1 and MAdCAM-1 on the apical surface of choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier, implies a previously unappreciated function of this barrier in the immunosurveillance of the CNS.     

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high K_m) for the substrate and by decreasing the number of transporter molecules (low V_max) on the colon luminal surface. The decreased transport activity at the CAM was associated with down‐regulation of the proton‐coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. J. Cell. Physiol. 226: 579–587, 2011. © 2010 Wiley‐Liss, Inc.     

The reduced folate carrier in L1210 murine leukemia cells is a 58-kDa protein

The reduced folate carrier (RFC1) is a major transporter for both natural reduced folates and antifolate chemotherapeutics. Using polyclonal antibodies targeted to epitopes at the loop between the sixth and seventh predicted transmembrane domains or the distal C-terminus, we were able to demonstrate by Western blot analysis that the molecular size of RFC1 expressed in murine leukemia L1210 cells is 58 kDa as predicted by the open reading frame of its cDNA. 46- and 38-kDa proteins detected only in plasma membrane preparations were proteolytic degradation products that appeared during membrane preparation or treatment with the conventional SDS-PAGE loading buffer. These data resolve discrepancies reported previously for the molecular size of RFC1.     

Placental folate transport during pregnancy

The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor alpha (FRalpha) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FRalpha, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRalpha but also RFC and heme carrier protein 1 (HCP1) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFRalpha, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FRalpha, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRalpha, RFC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.     

Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function

Intestinal folate transport has been well characterized, and rat small intestinal epithelial (IEC-6) cells have been used as a model system for the study of this process on the cellular level. The major intestinal folate transport activity has a low-pH optimum, and the current paradigm is that this process is mediated by the reduced folate carrier (RFC), despite the fact that this carrier has a neutral pH optimum in leukemia cells. The current study addressed the question of whether constitutive low-pH folate transport activity in IEC-6 cells is mediated by RFC. Two independent IEC-6 sublines, IEC-6/A4 and IEC-6/PT1, were generated by chemical mutagenesis followed by selective pressure with antifolates. In IEC-6/A4 cells, a premature stop resulted in truncation of RFC at Gln⁴²⁰. A green fluorescent protein (GFP) fusion with the truncated protein was not stable. In IEC-6/PT1 cells, Ser¹³⁵ was deleted, and this alteration resulted in the failure of localization of the GFP fusion protein in the plasma membrane. In both cell lines, methotrexate (MTX) influx at neutral pH was markedly decreased compared with wild-type IEC-6 cells, but MTX influx at pH 5.5 was not depressed. Transient transfection of the GFP-mutated RFC constructs into RFC-null HeLa cells confirmed their lack of transport function. These results indicate that in IEC-6 cells, folate transport at neutral pH is mediated predominantly by RFC; however, the folate transport activity at pH 5.5 is RFC independent. Hence, constitutive folate transport activity with a low-pH optimum in this intestinal cell model is mediated by a process entirely distinct from that of RFC. folic acid; folate absorption; methotrexate     

Xenopus reduced folate carrier regulates neural crest development epigenetically

Folic acid deficiency during pregnancy causes birth neurocristopathic malformations resulting from aberrant development of neural crest cells. The Reduced folate carrier (RFC) is a membrane-bound receptor for facilitating transfer of reduced folate into the cells. RFC knockout mice are embryonic lethal and develop multiple malformations, including neurocristopathies. Here we show that XRFC is specifically expressed in neural crest tissues in Xenopus embryos and knockdown of XRFC by specific morpholino results in severe neurocristopathies. Inhibition of RFC blocked the expression of a series of neural crest marker genes while overexpression of RFC or injection of 5-methyltetrahydrofolate expanded the neural crest territories. In animal cap assays, knockdown of RFC dramatically reduced the mono- and trimethyl-Histone3-K4 levels and co-injection of the lysine methyltransferase hMLL1 largely rescued the XRFC morpholino phenotype. Our data revealed that the RFC mediated folate metabolic pathway likely potentiates neural crest gene expression through epigenetic modifications.     

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.     

Structural basis for recognition and transport of folic acid in mammalian cells

Structural studies on mammalian vitamin transport lag behind other metabolites. Folates, also known as B9 vitamins, are essential cofactors in one-carbon transfer reactions in biology. Three different systems control folate uptake in the human body; folate receptors function to capture and internalise extracellular folates via endocytosis, whereas two major facilitator superfamily transporters, the reduced folate carrier (RFC; SLC19A1) and proton-coupled folate transporter (PCFT; SLC46A1) control the transport of folates across cellular membranes. Targeting specific folate transporters is being pursued as a route to developing new antifolates with improved pharmacology. Recent structures of the proton-coupled folate transporter, PCFT, revealed key insights into antifolate recognition and the mechanism of proton-coupled transport. Combined with previously determined structures of folate receptors and new predictions for the structure of the RFC, we are now able to develop a structure-based understanding of folate and antifolate recognition to accelerate efforts in antifolate drug development.     

A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRα cDNA was transfected into novel PCFT⁺ and PCFT^– HeLa sublines. FRα was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT^– cells. Cotransfection of FRα and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRα alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRα-mediated transport into the cytosol. FRα and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRα-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.Loss of function mutations of the proton-coupled folate transporter (PCFT),² which functions optimally at low pH, are the molecular basis for the autosomal recessive disorder, hereditary folate malabsorption (HFM) (1–4). Infants present with this disorder several months after birth with marked folate deficiency anemia, hypogammaglobulinemia with immune deficiency and infections, neurological deficits, and often seizures (5). PCFT is highly expressed at the apical brush-border membrane of the duodenum and proximal jejunum (6–9) where the pH at the microclimate of the surface of this epithelium is low (pH 5.8–6.0), and folates are absorbed (1, 7, 10, 11). Hence, the failure to absorb folates in the absence of this transporter in HFM is expected. However, PCFT expression, and its associated folate transport activity at low pH, is observed in many tissues where the transport interface is presumed to be at pH 7.4 (12). Of particular interest is the mechanism by which PCFT mediates transport of folates into the central nervous system (CNS) where this transporter is expressed in brain and choroid plexus (1, 7, 13). Transport into the CNS is impaired in patients with HFM who have very low cerebrospinal fluid (CSF) folate levels and marked reversal of the blood:CSF folate gradient which is normally 2–3:1 (5).Folates are also transported into cells by a receptor-mediated process. Folate receptor-α (FRα) is anchored to cell membranes via a glycosylphosphatidylinositol domain. Uptake begins with folate binding to receptor at the cell surface followed by invagination of the membrane and the formation of endosomes that traffic along microtubules to a perinuclear compartment before returning to the plasma membrane (14–16). During transit in the cytoplasm, endosomes acidify to a pH of ∼6.0–6.5 (17), folate is released from the receptor and exported from the intact endosome into the cytoplasm. This putative exporter was shown to require a trans-endosomal pH gradient (18–20).The current report addresses the hypothesis that PCFT is an endosomal folate exporter and thereby plays a role in FRα-mediated endocytosis (1, 2, 21, 22), that the ubiquitous expression of PCFT in mammalian tissues may be related to this function, and that loss of this function may be a basis for the low CSF folate levels in HFM. The experimental approach utilized a series of HeLa sublines, developed in this laboratory, in which constitutive expression of FRα is negligible. HeLa R5 cells lack reduced folate carrier (RFC) function due to a genomic deletion of this gene (23). A derivative of R5 cells, HeLa R1-11 cells lack, in addition, PCFT expression, while an R1-11 revertant re-expresses PCFT (24). The impact of PCFT on FRα-mediated endocytosis, achieved by transfection of the receptor into these cell lines, was assessed under conditions in which there was negligible PCFT-mediated transport directly across the plasma membrane into cells.     

Expression of RFC/SLC19A1 is associated with tumor type in bladder cancer patients

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.     

Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations. (i) Efflux of intracellular TPP was increased in cells with high expression of RFC1. (ii) Methotrexate inhibits TPP influx. (iii) TPP competitively inhibits methotrexate influx. (iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin. The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated.     

Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain.

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the alpha(1)-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:1255-1263, 1999)     

Identification of the Minimal Functional Unit of the Homo-oligomeric Human Reduced Folate Carrier

The reduced folate carrier (RFC) is the major transport system for folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).     

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.