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