Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

Hou, Zhanjun; Desmoulin, Sita Kugel; Etnyre, Erika; Olive, Mary B.; Hsiung, Benjamin; Cherian, Christina; Wloszczynski, Patrick A.; Moin, Kamiar; Matherly, Larry H.

doi:10.1074/jbc.m111.306860

Cited by 35 publications

(79 citation statements)

References 59 publications

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“…Nonetheless, in BN/PAGE conditions both monomeric proteins exhibit a mobility corresponding to an apparent molecular mass of~200 kDa when soluble markers are used for comparison [49]. In a previous study, PCFT's electrophoretic mobility in BN/PAGE conditions was predicted from comparison with soluble markers and the major band was determined to correspond to 190 kDa [50]. This is remarkably similar to the apparent mass obtained for 70 or 90 kDa GlyTs which may indicate that these 12 transmembrane span transporters have a comparable mobility under BN/PAGE conditions.…”

Section: Discussionmentioning

confidence: 99%

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The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

et al. 2013

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Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [ 3 H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitoninsolubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT 3A receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.

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Section: Discussionmentioning

confidence: 99%

“…A published report concluded that PCFT expressed heterologously in mammalian cells is homo-oligomeric [50]. Importantly, whole cell lysates were used in that study for crosslinker studies with the cysteine-specific reagent MTS-1-MTS.…”

Section: Discussionmentioning

confidence: 99%

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

et al. 2013

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“…The tissues were formalin-fixed and paraffin-embedded. The tissue microarrays were deparafinized, rinsed, microwaved, and incubated with polyclonal antibody to human PCFT raised in rabbits (Hou et al, 2012). The serum was purified using a peptide affinity column synthesized from Affi-Gel 10 (BioRad, Richmond, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Membrane proteins (30 mg) were electrophoresed on 7.5% polyacrylamide gels with SDS (Laemmli, 1970) and transferred to polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA) (Matsudaira, 1987). To detect PCFT, human PCFT-specific polyclonal antibody raised in rabbits to a carboxyl termini peptide (Hou et al, 2012) was used, with IRDye800CW-conjugated goat anti-rabbit IgG secondary antibody (LICOR Biosciences, Omaha, NE). Membranes were scanned and densitometry was performed with an Odyssey infrared imaging system (LICOR Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates

et al. 2016

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Pemetrexed (PMX) is a 5-substituted pyrrolo [2,3-d]pyrimidine antifolate used for therapy of nonsquamous nonsmall cell lung cancer (NS-NSCLC). PMX is transported by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Unlike RFC, PCFT is active at acidic pH levels characterizing the tumor microenvironment. By real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, PCFT transcripts and proteins were detected in primary NS-NSCLC specimens. In six NS-NSCLC cell lines (A549, H1437, H460, H1299, H1650, and H2030), PCFT transcripts and proteins were detected by real-time RT-PCR and western blots, respectively. 6-Substituted pyrrolo [2,3-d] H]C2 transport and decreased growth inhibition by C1 and C2, and to a lesser extent by PMX. In vivo efficacy of C1 was seen toward H460 tumor xenografts in severe-combined immunodeficient mice. Our results suggest that 6-substituted pyrrolo [2,3-d] pyrimidine thienoyl antifolates offer significant promise for treating NS-NSCLC by selective uptake by PCFT.

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“…For detecting total immunoreactive hPCFT and hRFC proteins on polyvinylidene difluoride membranes, hPCFTor hRFC-specific polyclonal antibodies raised in rabbits to carboxyl termini hPCFT or hRFC (Wong et al, 1998;Hou et al, 2012) peptides and an IRDye800CW-conjugated goat anti-rabbit IgG secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) were used. For detecting HA-tagged proteins (i.e., R5-RFC HA ), HA-specific mouse monoclonal antibody (Covance Research Products, Princeton, NJ) and an IRDye800CW-conjugated goat anti-mouse IgG secondary antibody (Rockland Immunochemicals) were used.…”

Section: Methodsmentioning

confidence: 99%

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

Desmoulin

Wang

Polin³

et al. 2012

Mol Pharmacol

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Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

Cited by 35 publications

References 59 publications

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

Targeting Nonsquamous Nonsmall Cell Lung Cancer via the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]Pyrimidine Thienoyl Antifolates

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

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