title: Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6.
abstract: 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 (G(93)XXXG(97)) and TMD4 (G(155)XXXG(159)). 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 [(3)H]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.
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A.
authors: Mike R
authors: MR
authors: Wilson
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A.
authors: Sita
authors: S
authors: Kugel
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A.
authors: Jenny
authors: J
authors: Huang
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A.
authors: Lucas J
authors: LJ
authors: Wilson
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A.
authors: Patrick A
authors: PA
authors: Wloszczynski
authors: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021 P.R. China.
authors: Jun
authors: J
authors: Ye
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A. Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, 48201 U.S.A. houz@karmanos.org matherly@karmanos.org.
authors: Larry H
authors: LH
authors: Matherly
authors: Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, 48201 U.S.A. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, 48201 U.S.A. houz@karmanos.org matherly@karmanos.org.
authors: Zhanjun
authors: Z
authors: Hou
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D058979:Proton-Coupled Folate Transporter / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human
keywords: anti-folate; cross-linking; folate; oligomerization; proton-coupled folate transporter; transporter