The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1–11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1–11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1–11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

The human proton-coupled folate transporter (hPCFT) is expressed in human solid tumors and is active at pHs characterizing the human microenvironment. hPCFT has 459 amino acids with 12 transmembrane domains and cytosolic N- and C-termini. Recent attention has focused on exploiting hPCFT for targeting solid tumors with novel cytotoxic antifolates (Kugel Desmoulin et al., Cancer Biol Ther 13: 1355-1373, 2012). hPCFT forms homo-oligomers with functional significance (Hou et al., J Biol Chem 287: 4982-4995, 2012). The hPCFT primary sequence includes GXXXG motifs in transmembrane domain (TMD) 2 (G93XXXG97) and 4 (G155XXXG159) that are analogous to dimerization motifs in other amphipathic proteins. To investigate the role of these GXXXG motifs in hPCFT oligomerization, we mutated Gly to Leu to obtain single (G93L, G97L, G155L, G159L) or multiple (G93L/G97L, G155L/G159L, G93L/G97L/G155L/G159L) mutants. Only with the G159L and G93L mutants, was [3H]methotrexate transport at pH 5.5 substantially preserved. Transport activity correlated closely with surface hPCFT levels by sulfo-NHS-SS-biotin labeling and Western blots. When hemagglutin (HA)-tagged G-to-L hPCFT mutants were co-expressed with FLAG-His10-tagged wild-type (WT) hPCFT in hPCFT-null HeLa cells, both mutant and WT proteins were retained on nickel affinity columns, suggesting that the hPCFT GXXXG motifs are not involved in protein oligomerization. This was substantiated by fluorescent resonance energy transfer (FRET) with N-terminal YFP-tagged and C-terminal CFP-tagged hPCFT constructs ectopically expressed in hPCFT-null HeLa cells. The GXXXG motifs in hPCFT are important for proper hPCFT folding and intracellular trafficking. A functional Cys-less (CL) hPCFT construct was generated in which the 7 Cys residues were replaced by Ser. Individual Cys insertion mutants were generated in which Cys was inserted from positions 115-137 in TMD3, and from positions 213-236 in TMD6. Constructs were transfected into hPCFT-null HeLa cells, followed by cross-linking with MTS-6-MTS (TMD3) or MTS-1-MTS (TMD6). Cross-links were detected for V118, A128, V132, Q136 and L137 in TMD 3, and for W213C, A227C, F228C, L234C, K235C, and E236C in TMD6, establishing proximities between vicinal TMDs in separate hPCFT monomers. These results strongly imply that TMD3 and TMD6 provide critical polypeptide interfaces for the formation of hPCFT oligomers. Identification of structural motifs or domains involved in hPCFT oligomerization may lead to novel approaches for therapeutically “rescuing” functionally impaired hPCFT mutants, or enhancing surface expression of hPCFT in tumors treated with hPCFT-selective antifolates. Key words: proton-coupled folate transporter, folate, oligomerization, cross-linking, transporter. Citation Format: Zhanjun Hou, M. Roy Wilson, Lucas Wilson, Sita Kugel Desmoulin, Jenny Huang, Larry H. Matherly. Identification of structural determinants of human proton-coupled folate transporter oligomerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 783. doi:10.1158/1538-7445.AM2014-783