The transcriptional and post-transcriptional controls that contribute to tissue-specific expression of RFC have been largely defined (4
). While there have been extensive studies of high frequency sequence polymorphisms in the hRFC coding and noncoding regions in relation to a variety of pathological states (4
), these associations remain controversial. The recent production of a “humanized” RFC mouse in which the mouse RFC gene is functionally replaced by the hRFC gene locus (198
) holds promise for extending in vitro
findings of hRFC regulation and function including polymorphic hRFC gene variants to a clinically relevant in vivo
model. This will allow correlations with dietary folate status and biological manifestations of folate deficiency.
Structure-function studies of hRFC have provided substantive insights into its membrane topology, N-glycosylation, important domains and amino acids, and three dimensional helix packing associations (4
). Recent studies indicate that there are higher order oligomers (e.g., dimers) of hRFC (Z. Hou and L.H. Matherly, unpublished), as has been observed for other transporters (199
). Clarification of RFC oligomeric character is especially important to understanding carrier structure and function, including the mechanism of concentrative folate transport. The existence of homo-oligomeric hRFC may also play an important role in antifolate resistance through potential “dominant-negative” interactions between mutant and wild-type hRFCs that result in alterations in function and/or trafficking defects.
Since PCFT was only recently discovered, there is only scant information on the structural properties of this transporter. A predicted secondary structure was described and localization of the N- and C-termini and the first extracellular loop was verified (66
). Studies are ongoing to identify key amino acid residues that are determinants of substrate binding, proton-coupling, and carrier mobility. Future studies will emphasize helix packing and tertiary structure using approaches such as cysteine-scanning mutagenesis and accessibility methods (35
). Of course, the finding of higher order hRFC oligomers raises the intriguing possibility that oligomeric PCFT may be important, as well. All these studies of PCFT structure and function would be enhanced considerably by the availability of a three dimensional structural model of this transporter. The extrapolation of a prokaryotic model based upon the GlpT crystal structure to PCFT is appealing (136
) since GlpT is also proton-coupled although, unlike PCFT, this is a proton antiporter (201
). Extrapolation of this and other models to PCFT or other transporters will require rigorous experimental verification, as noted above for RFC.
Since folate deficiency is associated with increased risk of colorectal cancer, deciphering the role of RFC and PCFT in the delivery of folates to the large intestine and their regulation at this site, within the context of dietary and environmental factors, will be of considerable importance. These studies need to take into consideration the recent paradigm that folate deficiency results in an increased frequency of oncogenesis, but once cancers are formed, excess folates may accelerate tumor growth (202
). For RFC, causal associations with loss of carrier function and colorectal cancer were described in mouse models (204
The hypothesis that PCFT is required for FR function, by serving as a route of export from acidified endosomes, broadens the potential biological importance of this carrier. Clarification of this issue could establish a critical role for PCFT in the transport of folates across the blood-choroid plexus-barrier and clarify why transport into this compartment is impaired in HFM. Additional information is also required to clarify the functional role of PCFT in other tissues in which it is highly expressed, particularly those expected to harbor a neutral pH milieu. This relates, in particular, to the extent to which PCFT mediates folate transport from the hepatic portal vein across the sinusoidal membrane to hepatic cells, reabsorption in the proximal renal tubule, transport across the placenta to the fetus, and transport across the choroid plexus and into brain parenchyma at the blood-brain-barrier. These questions will be best addressed by studies on a PCFT-null mouse.
The role that PCFT plays in antifolate delivery to tumor cells requires further exploration. PCFT has a high affinity for pemetrexed and, when transfected into tumor cells, selectivity augments the drug’s growth inhibitory activity without a salutary effect on other antifolates such as MTX, PT523 or raltitrexed which have a much lower affinity for PCFT at physiological pH (67
). It will be important to determine whether PCFT might contribute more to the activity of pemetrexed, or at all to the activities of other antifolates, when transport occurs within solid tumors in vivo
where cells are hypoxic, the microenvironment is somewhat acidic, and PCFT would be expected to operate more efficiently (206
). Likewise, because of the high Km
s for antifolate transport at physiological pH, it will be important to determine the role PCFT plays in the delivery of antifolates at the high blood levels achieved in clinical regimens under conditions in which RFC is saturated.
Finally, there continues to be an ongoing interest in the development of new-generation antifolates. Antifolate thymidylate synthase inhibitors were designed with very low affinity for RFC but very high affinity for FRα, so as to minimize toxicity due to uptake via RFC which is ubiquitously expressed, and to maximize uptake via FRs that are more selectively expressed in certain tumors (209
). An analogous strategy was very recently described for a series of highly potent FR-targeted antifolate GARFT inhibitors (212
). It will be of particular interest to determine whether the activities of these novel FR-targeted agents will be limited by their efficacies as substrates for PCFT which may be required for their optimal export from endosomes during the endocytic cycle.