Structure and function of the reduced folate carrier a paradigm of a major facilitator superfamily mammalian nutrient transporter

Abstract

Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurological disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathological conditions. This article focuses on the biology of the membrane transport system termed the "reduced folate carrier" or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiological or developmental consequences. For chemotherapeutic antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its N-glycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial major facilitator superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.

Figure 1. Transport substrates for the reduced folate carrier

Structures are shown for folate and antifolate substrates for RFC.

Figure 2. Topologic model for hRFC showing conserved residues between 7 species

Topology model for hRFC, depicting 12 TMDs, internally oriented N and C termini, an externally orientated N-glycosylation site at Asn-58 and a cytosolic loop connecting TMDs 6 and 7. Amino acids conserved between RFCs from different species as summarized in Figure 3 are depicted as black circles.

Figure 3. Species homologies for RFC proteins

Genbank accession numbers are: Homo sapiens (human) NP 001069921; Pan troglodytes (chimpanzee) XP 001157360; Gallus gallus (chicken,) NP 001006513; Danio rerio (zebrafish) XP 687261; Bos taurus (cow)NP 001069921; Rattus norvegicus (Norway rat) NP 001030309; Cricetulus griseus (Chinese hamster) U17566; Mus musculus (mouse) NP 112473; Xenopus laevis (African clawed frog) NM 001092530.

Figure 3. Species homologies for RFC proteins

Genbank accession numbers are: Homo sapiens (human) NP 001069921; Pan troglodytes (chimpanzee) XP 001157360; Gallus gallus (chicken,) NP 001006513; Danio rerio (zebrafish) XP 687261; Bos taurus (cow)NP 001069921; Rattus norvegicus (Norway rat) NP 001030309; Cricetulus griseus (Chinese hamster) U17566; Mus musculus (mouse) NP 112473; Xenopus laevis (African clawed frog) NM 001092530.

Figure 4. Proposed 3-D models of hRFC based on solved crystal structures of LacY and GlpT and SCAM analysis, and the hypothesized substrate binding site of hRFC

A three dimensional hypothetical model for hRFC is presented based on structure alignments between hRFC and LacY and GlpT and fine-tuned based on experimental SCAM data. Modeling was performed with the Modeller 8v1 auto mode (Marti-Renom, 2000). All models were drawn by PyMol (DeLano, 2002). Panel A depicts a side view of hRFC for which the extended C-terminal segment is truncated at Lys-479. TMDs 1, 2, 4, and 5 of the N-terminal region and TMDs 7, 8, 10, and 11 of the C-terminal region are hypothesized to be involved in formation of the hydrophilic cavity for anionic substrate binding (colored as black). TMDs 3, 6, 9, and 12 are likely buried in the lipid bilayer and do not directly participate in substrate binding (colored as grey). Panel B depicts a cytosolic view of only the TMD segments (numbered 1-12 as in Figure 2) of the hRFC molecule so that the order of helix packing can be easily seen. TMD shading is the same as in Panel A. Panel C shows an enhanced view of the hypothetical substrate binding site, including Lys-411, Ser-313, Tyr-281, and Arg-373, as described in the text. Other residues that may contribute to the substrate binding pocket are also shown and include Arg-133, Ile-134, Ala-135, Tyr-136, and Ser-138. The physical distances between α carboxyl groups of Lys-411, Ser-313, Tyr-281 and Arg-373 are shown in Å. Adapted from Hou et al. (2006).