Abstract 1524: Identification of a critical oligomerization domain of the human reduced folate carrier

Abstract The human reduced folate carrier (hRFC) is the major membrane transport system for folates in human tissues and tumors. Losses of hRFC expression and function may be causal in folate deficiencies that result in a range of conditions including cancer. hRFC is also a major transporter for antifolate drugs such as methotrexate (Mtx), pemetrexed, and raltitrexed used for cancer, and losses of hRFC surface expression and function are common modes of antifolate drug resistance. Monomeric hRFC is a single 591 amino acid polypeptide, composed of 12 transmembrane domain (TMD) segments with cytosolic N- and C- termini, a N-glycosylation site, and a large connecting loop domain between TMDs 6 and 7. hRFC can be expressed as “half molecules” (HMs) from separate TMD1-6 and TMD7-12 HM constructs to restore wild type (wt) carrier function. Our recent studies establish that hRFC exists as homo-oligomers that are important to intracellular trafficking from the endoplasmic reticulum (ER) to the plasma membrane, and potentially to carrier function. Mutant hRFCs poorly expressed at the cell surface interfere with intracellular trafficking of wt hRFC from ER, by virtue of formation of wt-mutant oligomers. To begin to identify structural determinants of hRFC oligomerization, we used hRFC HMs and a panel of “partial molecule” (PM) constructs (TMD1-5, TMD1-4, TMD1-3, TMD1-2, TMD1,3-6, TMD2-6, TMD8-12, TMD9-12, TMD10-12 and TMD11-12) prepared from HM hRFCs. HMs and PMs were transiently co-expressed with wt hRFC in hRFC-null R5 HeLa cells. Transport activity was assayed with [3H]Mtx, whereas expression levels of full-length wt, and HM and PM hRFCs were assayed on westerns with hRFC- or epitope- (HA- or Myc) specific antibodies. Whereas co-expression of TMD7-12 with wt hRFC inhibited activity and surface expression of wt hRFC greater than 90% resulting from formation of wt-HM oligomers, the impact of the PMs and TMD1-6 HM on wt hRFC was nominal. This strongly suggested that a critical oligomerization domain involves amino acids in or flanking TMD7 of hRFC. We further localized this stretch of amino acids within TMD7 by co-expressing a refined series of eight TMD7-12 deletion mutants in which 5 residues from A252 to W292 in or flanking TMD7 were sequentially deleted. Deletions from L267 to Y286 within TMD7 were highly specific in effecting loss of wt hRFC surface expression and function. Our results strongly suggest that a critical hRFC oligomerization domain maps within TMD7. The implications of our findings to hRFC pharmacology and physiology are immense in terms of identifying potential determinants of decreased hRFC expression resulting in loss of folate and antifolate membrane transport, and possible strategies for therapeutically modulating hRFC homo-oligomerization in relation to cancer etiology or therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1524.