Abstract 782: Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important

Abstract

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with a twelve transmembrane domain (TMD) structure. Human PCFT (hPCFT) is highly expressed in many solid tumors and has an acidic pH optimum, suggesting its high activity in the acidic tumor microenvironment. Our recent studies have focused on exploiting PCFT for targeting solid tumors with novel cytotoxic antifolates. Predicted topology models suggest that the loop domain between TMDs 2 and 3 resides in the cytosol. However, mutations in TMD2-3 loop residues Asp109 or Arg113 result in loss of PCFT activity, suggesting an important role in transport activity. Additionally, TMD2 may itself form a part of the substrate binding domain, suggesting functional importance for TMD2 residues. Using site-directed mutagenesis, we replaced each of the seven cysteine (cys) residues of hPCFT with serine, creating functional “cys-less” PCFT for use in substituted cys accessibility methods (SCAM). We then used site-directed mutagenesis to create 34 single cys mutants spanning TMD2 and the TMD 2-3 loop region (positions 85-118) in a cys-less PCFT background. These mutants were transfected into R1-11 HeLa cells, which express no endogenous folate transporters. We measured [3H]methotrexate uptake at pH 5.5 as a readout of PCFT activity and confirmed PCFT protein expression by western blotting. All 34 mutants were detected on westerns and 29 mutants were active, while W107C, D109C, G112C, R113C, and R114C were inactive. Using functionally active mutants, we performed pull-down assays with 2-aminoethyl methanethiosulfonate-biotin (MTSEA-biotin) and steptavidin beads, in order to determine the aqueous accessibility of each mutant. We found that mutants G93C, F94W, G105C, A106C, S110C, and L116C reacted with MTSEA-biotin, suggesting that these residues are aqueous-accessible. For G93C, F94C, and L116C, MTSEA-biotin labeling was inhibited by pemetrexed pretreatment, and MTSEA-biotin treatment inhibited transport activity of G93C and F94C. Our results suggest that the TMD 2-3 loop domain is aqueous accessible and must form a novel functionally important “reentrant loop” structure. Additionally, residues located in TMD2 (Gly93 and Phe94) appear to play a key role in substrate binding to PCFT. Better understanding of functional and structural determinants of PCFT membrane transport will be critical to the development of novel PCFT-selective antifolate drugs for targeting solid tumors. Citation Format: Michael R. Wilson, Zhanjun Hou, Larry H. Matherly. Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important. [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 782. doi:10.1158/1538-7445.AM2014-782