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Macrophage activation comprises a continuum of functional states critically determined by cytokine microenvironment. Activated macrophages have been functionally grouped according to their response to pro-Th1/proinflammatory stimuli [lipopolysaccharide, IFNgamma, granulocyte macrophage colony-stimulating factor (GM-CSF); M1] or pro-Th2/anti-inflammatory stimuli [interleukin (IL)-4, IL-10, M-CSF; M2]. We report that folate receptor beta (FRbeta), encoded by the FOLR2 gene, is a marker for macrophages generated in the presence of M-CSF (M2), but not GM-CSF (M1), and whose expression correlates with increased folate uptake ability. The acquisition of folate uptake ability by macrophages is promoted by M-CSF, maintained by IL-4, prevented by GM-CSF, and reduced by IFNgamma, indicating a link between FRbeta expression and M2 polarization. In agreement with in vitro data, FRbeta expression is detected in tumor-associated macrophages (TAM), which exhibit an M2-like functional profile and exert potent immunosuppressive functions within the tumor environment. FRbeta is expressed, and mediates folate uptake, by CD163(+) CD68(+) CD14(+) IL-10-producing TAM, and its expression is induced by tumor-derived ascitic fluid and conditioned medium from fibroblasts and tumor cell lines in an M-CSF-dependent manner. These results establish FRbeta as a marker for M2 regulatory macrophage polarization and indicate that folate conjugates of therapeutic drugs are a potential immunotherapy tool to target TAM.

The glycosyl-phosphatidylinositol anchored folate receptor (FR) mediates selective delivery of a broad range of experimental drugs to the receptor-rich tumors, but molecular mechanisms controlling FR internalization have not been adequately studied. FR quantitatively recycles between the cell surface and endocytic compartments via a Cdc42-dependent pinocytic pathway. Protein kinase C (PKC) activators including diacylglycerol and phorbol ester have previously been reported to increase the proportion of FR on the cell surface. Here we identify the alpha-subtype of PKC as the mediator of phorbol ester action on FR recycling and provide evidence that activated PKCalpha is recruited to FR-rich membrane microdomains where, in association with its receptor RACK1, it inhibits FR internalization; the activation state of Cdc42 remains unaltered. We also show that the PKC substrate, annexin II, is required for FR internalization. The studies clarify a molecular mechanism for the regulation of FR recycling through PKC which could potentially be exploited for effective drug delivery.

Objective: To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX)., Methods: Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells., Results: Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells., Conclusion: Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

Estradiol (E2) acts through the estrogen receptor (ER) to downregulate many genes, and tamoxifen (Tam) largely reverses this repression but the underlying mechanisms are unclear. Repression of the folate receptor (FR)-alpha P4 core promoter by ER is enhanced by E2 and reversed by Tam. This effect was unaffected by inhibition of new protein synthesis and required the E/F and the DNA-binding domains of ER without direct binding of ER to DNA. The repression by E2/ER was not specific for either Sp1 or TATA elements but was loosely selective for the initiator and flanking sequence. Insertion of a response element or a relatively strong Sp1 cluster to recruit ER upstream of the core promoters caused a switch to activation by E2/ER that was inhibited by Tam. In nuclear extracts, association of ER with a biotinylated core promoter fragment was promoted by E2 but Tam blocked this effect. Repression/de-repression of the P4 promoter and endogenous FR-alpha expression by E2/Tam required SMRT and/or NCoR. ER associated with the chromosomal P4 promoter and SMRT and NCoR associated with it in an ER-dependent manner; these associations were favored by E2 but disrupted by Tam, in the short term, without changes in ER expression. TAFII30 was required for optimal P4 promoter activity and for the repressive association of ER. E2 may thus maintain a low transcriptional status of genes by favoring direct TAFII30-dependent association of ER with the core promoter in a co-repressor complex containing SMRT and/or NCoR; this repression is overridden in target genes containing an upstream element that strongly recruits ER. In addition to suppressing the activation of classical E2 target genes, Tam may upregulate genes by passively dissociating the ER co-repressor complex.

Targeted drug delivery has the potential to improve the efficacy of a therapeutic agent while reducing its side effects. The folate receptor type beta (FR-beta) is a cell surface marker selectively expressed in the leukemic cells of approximately 70% of acute myeloid leukemia (AML) patients. Upregulation of FR-beta may also be selectively induced in AML cells by treatment with all-trans-retinoic acid (ATRA). In this study, the role of formulation composition in FR-targeted liposomal doxorubicin (DOX) delivery to AML cells was investigated. Liposomal formulations with a variable percentage of folate-polyethylene glycol distearoyl phosphatidylethanolamine (f-PEG-DSPE) were synthesized and evaluated for FR-beta-targeted DOX delivery in MV4-11 AML cells in vitro and for their pharmacokinetic properties in vivo. The formulation containing 0.5 mol % f-PEG-DSPE exhibited the highest efficiency of cellular uptake and in vitro cytotoxicity, as well as a long systemic circulation time in mice. In MV4-11 cells, the binding and cytotoxicity of FR-targeted liposomal DOX based on this formulation was also enhanced by ATRA-induced FR-beta upregulation.

For over a decade the folate receptor has been intensively investigated as a means for tumor-specific delivery of a broad range of experimental therapies including several conceptually new treatments. Despite a few set backs in clinical trials, the literature is replete with encouraging in vitro and pre-clinical studies of gynecological and other tumors and more therapeutic approaches are ready for clinical testing. Recent studies have added myelogenous leukemias to the list of candidate cancers for FR-targeted therapies. Each approach faces unique challenges in translation that could be addressed through a mechanistic understanding of the function and expression of the receptor in the appropriate experimental systems and by improvements in the technology. This review discusses FR in the context of positive recent developments in broad areas of FR-targeted therapy and attempts to highlight its potential and the anticipated challenges.

The folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML), owing particularly to its selective up-regulation in the leukemic cells by all-trans retinoic acid (ATRA). Here we show, using KG-1 and MV4-11 AML cells and recombinant 293 cells, that the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VPA), and FK228 potentiated ATRA induction of FR-beta gene transcription and FR-beta mRNA/protein expression. ATRA and/or TSA did not induce de novo FR synthesis in any of a variety of FR-negative cell lines tested. TSA did not alter the effect of ATRA on the expression of retinoic acid receptor (RAR) alpha, beta, or gamma. Chromatin immunoprecipitation assays indicate that HDAC inhibitors act on the FR-beta gene by enhancing RAR-associated histone acetylation to increase the association of Sp1 with the basal FR-beta promoter. Under these conditions, the expression level of Sp1 is unaltered. A decreased availability of putative repressor AP-1 proteins may also indirectly contribute to the effect of HDAC inhibitors. Finally, FR-beta selectively mediated growth inhibition by (6S) dideazatetrahydrofolate in a manner that was greatly potentiated in AML cells by ATRA and HDAC inhibition. Therefore, the combination of ATRA and innocuous HDAC inhibitors may be expected to facilitate selective FR-beta-targeted therapies in AML.

The expression patterns of folate receptor (FR) isoforms, alpha and beta, in normal and malignant male and female reproductive tissues is described. The significance of the receptor in reproductive and developmental physiology is discussed. The potential value of the receptor expressed in malignant tissues including ovarian and endometrial cancers as a diagnostic marker and a therapeutic target is reviewed. Finally, the various transcriptional and post-transcriptional mechanisms that govern the tissue/tumor-specificity of the receptor and its regulation by folate and steroid receptor ligands are described; the potential value of this knowledge in developing better methods for the early detection and treatment of certain cancers is discussed.

The utility of the folate receptor (FR) type alpha, in a broad range of targeted therapies and as a diagnostic serum marker in cancer, is confounded by its variable tumor expression levels. FR-alpha, its mRNA and its promoter activity were coordinately up-regulated by the glucocorticoid receptor (GR) agonist, dexamethasone. Optimal promoter activation which occurred at <50 nmol/L dexamethasone was inhibited by the GR antagonist, RU486, and was enhanced by coactivators, supporting GR mediation of the dexamethasone effect. The dexamethasone response of the FR-alpha promoter progressed even after dexamethasone was withdrawn, but this delayed effect required prior de novo protein synthesis indicating an indirect regulation. The dexamethasone effect was mediated by the G/C-rich (Sp1 binding) element in the core P4 promoter and was optimal in the proper initiator context without associated changes in the complement of major Sp family proteins. Histone deacetylase (HDAC) inhibitors potentiated dexamethasone induction of FR-alpha independent of changes in GR levels. Dexamethasone/HDAC inhibitor treatment did not cause de novo FR-alpha expression in a variety of receptor-negative cells. In a murine HeLa cell tumor xenograft model, dexamethasone treatment increased both tumor-associated and serum FR-alpha. The results support the concept of increasing FR-alpha expression selectively in the receptor-positive tumors by brief treatment with a nontoxic dose of a GR agonist, alone or in combination with a well-tolerated HDAC inhibitor, to increase the efficacy of various FR-alpha-dependent therapeutic and diagnostic applications. They also offer a new paradigm for cancer diagnosis and combination therapy that includes altering a marker or a target protein expression using general transcription modulators.

Strategic modalities of drug delivery have the potential to greatly improve the therapeutic efficacy of available drugs in acute myelogenous leukaemia (AML). Folate receptor (FR) type beta is selectively expressed on the surface of approximately 70% of AMLs. Increased FR-beta expression in these cells can be induced by all-trans retinoic acid (ATRA) and other retinoid compounds in the absence of terminal differentiation or cell growth inhibition. An apparent post-transcriptional modification prevents FR-beta in normal haematopoietic cells from binding folate, in contrast to AML cells. FR-beta may, therefore, be used as a target for the selective delivery of chemotherapeutic drugs to AML cells; this treatment modality appears to be particularly efficacious when administered in conjunction with retinoid-induction of FR-beta. FR-targeted liposomal drug delivery can also bypass the P-glycoprotein (P-gp)-mediated drug efflux pump commonly associated with multiple drug resistance in AML. The rationale and merits of this novel experimental treatment for AML and the current status of this research are provided.

The folate receptor (FR) type alpha is a promising target for diagnostic imaging agents and therapeutic intervention in major subtypes of gynecological malignancies; however, the receptor levels in the tumors are variable and are generally relatively low in estrogen receptor (ER)-positive tumors. Here we report that the FR-alpha gene promoter is repressed in the presence of 17beta-estradiol and derepressed by the antiestrogens tamoxifen and ICI 182780 in a promoter-specific and ER-alpha-dependent manner in carcinoma cell lines including HeLa (cervical carcinoma), BG-1 (ovarian carcinoma), and IGROV-1 (ovarian carcinoma). The ligand and ER dose response of the FR-alpha promoter and its time course paralleled those of a classical estrogen response element-mediated effect. Antiestrogens produced an ER-dependent increase of up to 36-fold in the expression of the endogenous FR-alpha gene. Deletion analysis and FR-alpha/SV40 promoter chimeras showed that the ER effect is mediated exclusively within the G/C-rich region in the TATA-less P4 promoter of FR-alpha; electrophoretic mobility shift analysis demonstrated interaction of ER at only one of three G/C-rich elements. ER-beta only modestly affected FR-alpha promoter activity but did not diminish the ER-alpha-mediated effects. The ER corepressor, SMRT, enhanced the repression by 17beta-estradiol/ER, but ER coactivators, including SRC family members, did not appreciably impact the ER ligand response. The results suggest that in ER+ tumors, FR-alpha expression is directly and actively suppressed and predict that a brief treatment with antiestrogens will boost FR-alpha expression by passive derepression, enhancing the efficacy of FR-targeted diagnostic and therapeutic applications. They also reveal novel aspects of gene repression by ER.

Folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML) owing particularly to its specific up-regulation in AML cells by all-trans retinoic acid (ATRA). Here we identify functional elements in the FR-beta gene and examine the molecular mechanism of transcriptional induction of FR-beta by ATRA. The basal promoter activity of FR-beta resulted from synergistic interaction between Sp1 and ets binding sites (EBSs) and repression by upstream AP-1-like elements, whose action required EBSs. A minimal promoter containing the Sp1 and ets elements was ATRA-responsive. The repressor elements bound Fos family proteins; association of the proteins with the repressor elements correlated negatively with FR-beta expression in peripheral blood neutrophils and monocytes and also in KG-1 (AML) cells grown in the absence or in the presence of ATRA. Furthermore, down-regulation of FR-beta in KG-1 cells treated with O-tetradecanoylphorbol 13-acetate (TPA) was accompanied by increased AP-1 binding to the repressor elements. From chromatin immunoprecipitation (ChIP) assays, the nuclear retinoic acid receptor alpha (RARalpha) associated with the Sp1 region, and RARs beta and gamma associated with the AP-1 and Sp1 regions; treatment of KG-1 cells with ATRA did not alter Sp1 binding but increased the association of RARalpha and decreased the association of RARs beta and gamma. ATRA also decreased RAR expression levels. The results suggest that the FR-beta gene is a target for multiple coordinate actions of nuclear receptors for ATRA directly and indirectly acting on a transcriptional complex containing activating Sp1/ets and inhibitory AP-1 proteins. The multiple mechanisms favor the prediction that ATRA will induce FR-beta expression in a broad spectrum of AML cells. Further, optimal FR-beta induction may be expected when all 3 RAR subtypes bind agonist.

The human folate receptor (hFR) type gamma gene is driven by a TATA-less promoter that uses a canonical Sp1 element for basal transcription. Using nuclear extract from 293 (human embryonic) cells, we mapped a second (non-canonical) Sp1 element to which Sp1 bound with a comparable affinity and which overlaps a functional ets binding site (EBS). Mutagenesis experiments revealed that the binding of ets to the EBS activates the promoter synergistically with Sp1 bound at the downstream site; however, binding of Sp1 to the EBS does not contribute to promoter activity. A further increase in Sp1 by inducible expression in recombinant 293 cells resulted in a small but significant decrease in the hFR-gamma promoter activity, but the decrease was abolished when the EBS was deleted from the promoter. In 293 cells, which do not express hFR-gamma, the Sp1 level was relatively high whereas in the hFR-gamma-positive HL60 leukemia cells, the Sp1 level was low and the EBS predominantly bound an ets protein. To account for the above observations, we propose a model in which when the Sp1 level is low, ets out competes Sp1 for binding to the EBS and synergistically enhances the hFR-gamma promoter activity by interacting with Sp1 bound at the canonical site whereas at higher levels, Sp1 represses the promoter by competitively inhibiting the binding of ets. As a partial extension of this model to the regulation of other ets activated genes, we show that Sp1 can predictably bind to a variety of ets elements including those responsive to Ets1 and Spi.1/Pu.1. A dual concentration-dependent action of Sp1 as an activator or a repressor offers a potential mechanism contributing to tissue-specific regulation of ets-dependent genes by Sp1.

The folate receptor type alpha (FR-alpha) is a promising tumor marker and target. Here, we investigate the mechanistic basis for the tumor specificity and vast overexpression of FR-alpha. Among representative FR-alpha-positive (HeLa and JAR) and FR-alpha-negative (MG63, Caki1, and HT3) cell lines, the transcription rates of the endogenous FR-alpha gene, as well as the FR-alpha promoter activity, were relatively weak and comparable, but the FR-alpha transcript was abundant only in total RNA and nuclear RNA from the FR-alpha-positive cells. Rous sarcoma virus (RSV) promoter-driven expression of the FR-alpha gene was 7 to 30 times greater in the FR-alpha-positive than in FR-alpha-negative cells, both at the protein and mRNA levels, independently of intron sequences. Through the use of chimeric FR-alpha/FR-beta cDNAs, the above pattern of FR-alpha expression was attributed to a 60-bp sequence in the FR-alpha open reading frame. This sequence element, when placed in the 5' untranslated region of RSV promoter-luciferase, decreased the reporter expression approximately 7- to 20-fold in FR-alpha-negative cells (MG63, Caki1, HT3, BG1, and MCF7) relative to FR-alpha-positive cells (HeLa, JAR, and JEG3). Substitution of this FR-alpha element in FR-beta increased the in vivo degradation rate of the transcript in the nuclei of MG63 cells but not in the nuclei of HeLa cells or in the cytosol of MG63 or HeLa cells. The results reveal an efficient mechanism by which a novel sequence element causes differential transcript degradation in the nucleus to ensure narrow tissue specificity for a gene (e.g., that for FR-alpha) whose transcription is weak and relatively nonselective. FR-alpha exhibited constitutive mRNA and protein synthesis during the cell cycle and a slow protein turnover, presumably ensuring a high steady-state level of the receptor in cells that could override the nuclear mRNA instability determinant.

Cholesterol and glycosphingolipid-rich membrane rafts, which are rich in GPI-anchored proteins and are distinct from caveolae, are believed to serve as platforms for signal transduction events and protein recycling. GPI-anchored proteins with diverse functions as well as caveolin may be recovered in a membrane fraction insoluble in cold non-ionic detergent. This study tests for possible heterogeneity in the protein composition of the lipid rafts and detergent-insoluble membrane complexes by examining the two GPI-anchored homologous human folate receptors (FR)-alpha and -beta, the GPI-anchored human placental alkaline phosphatase (PLAP), and caveolin (control) in transfected CHO cells. Both FR and PLAP showed the equal distribution of cell-surface vs. sequestered (recycling) protein typical of GPI-proteins. Quantitative affinity purification of detergent-insoluble complexes using biotinylated folate or specific antibodies demonstrated a strong association of the homologous FR-alpha and FR-beta in the same detergent-insoluble complex and separate complexes containing either PLAP or caveolin. Immunogold localization experiments using antibody crosslinking to produce larger aggregates of GPI-anchored proteins for visualization by electron microscopy also showed a clear separation between FR- and PLAP-rich membrane microdomains. Thus, even though functionally diverse and heterologous GPI-anchored proteins are known to share endocytic and recycling vesicles, they may be segregated in distinct lipid rafts on the basis of their ecto(protein) domains facilitating clustering, compartmentalization and homotypic protein interactions.

Up-regulation of folate receptor (FR) type-beta in acute myelogenous leukemia (AML) by all-trans retinoic acid (ATRA) and its restricted normal tissue distribution makes it a potential target for therapeutic intervention. The FR-beta in peripheral blood granulocytes was unable to bind folate and appeared to have a variant GPI membrane anchor, evident from its insensitivity to phosphatidylinositol-specific phospholipase C but not nitrous acid. Granulocyte FR-beta lacked mutations, and neither deglycosylation nor detergent solubilization restored folate binding. The posttranslational modification causing its nonfunctionality was evidently absent in FR-beta from AML cells from patient marrow, which bound folate. From flow cytometric analysis of 78 AML bone marrow specimens of different subtypes, 68% expressed FR-beta, most of which were also CD34+. In model cell lines that are FR - (KG-1a, L1210, and Chinese hamster ovary [CHO]) or FR + (KG-1, L1210 JF, and recombinant CHO-FR-beta), selective FR-mediated binding and cytotoxicity was obtained using folate-coated liposomes encapsulating fluorescent calcein (f-L-calcein) and doxorubicin (f-L-DOX), respectively, which could be blocked by 1 mM free folic acid. In the FR-beta-expressing KG-1 human AML cells, treatment with ATRA further increased this specificity. In mouse ascites leukemia models generated using L1210JF or KG-1 cells, increased median survival times were obtained with f-L-DOX treatment compared to nontargeted L-DOX. In the KG-1 model, ATRA treatment increased the cure rate with f-L-DOX from 10% to 60%. The above combined data from our 2 laboratories further support the feasibility and potential usefulness of selective ATRA-facilitated liposomal drug delivery in FR-beta + AMLs.

Folate receptor (FR) type beta is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-beta expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-beta expression in vitro in myeloid leukemia cells from patient marrow. FR-beta was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D(3), or transforming growth factor beta. ATRA did not induce FR-beta expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-beta expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-beta promoter-luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-beta promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)alpha, RARbeta, or RARgamma, it appears that the retinoid effect on FR-beta expression could be mediated by ligand binding to RARs alpha, beta, or gamma, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARalpha and RARgamma selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-beta expression. Thus, blocks in the RARalpha-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-beta expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-beta expression in myeloid leukemia cells refractory to retinoid differentiation therapy.

The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.

Background: The membrane-associated folate receptor (FR) type beta is elevated in the spleen in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). In this study, the authors investigated possible cell type and differentiation stage specificity of expression of FR-beta in normal and leukemic hematopoietic cells., Methods: An affinity-purified rabbit polyclonal antibody specific for FR-beta was employed for immunostaining representative bone marrow smears and peripheral blood smears from normal individuals and from a limited number of patients with various leukemias. Multiple samples of normal bone marrow and peripheral blood were analyzed for the expression of FR-beta and selected CD antigens by two- or three-color flow cytometry., Results: Of the morphologically identifiable cells, only neutrophils were positive for FR-beta. The leukemic blasts in CML patients showed expression of FR-beta with no apparent relation to the occurrence of the Philadelphia chromosome. Among acute nonlymphocytic leukemias, FR-beta was expressed in promyelocytic leukemia, in the myeloblast populations of myelomonocytic and erythroleukemias, and variably in M1/M2 AML. Neither the blasts of acute lymphocytic leukemia nor the more mature cells of chronic lymphocytic and hairy cell leukemias expressed FR-beta. The less differentiated FR-beta positive AML samples also were positive for CD34 and HLA-DR. Flow cytometric analysis of normal bone marrow and peripheral blood revealed low or insignificant coexpression of FR-beta with CD34, CD19, and CD3, whereas significant coexpression was observed with high levels of CD33, CD13, and CD11b; coexpression of FR-beta with CD14 was high in the immature bone marrow cells, comparable to that in myeloid cells, but relatively low in peripheral blood., Conclusions: The results of this study suggest a narrow expression pattern of FR-beta marking the neutrophilic lineage and the possibility of defining a subtype or subtypes of myeloid leukemia based on FR-beta expression. The identification of FR-beta positive leukemias and the absence of the receptor in normal CD34 positive cells may enable selective receptor-mediated targeting of leukemic cells.

The human folate receptor (hFR) type gamma and gamma' are constitutively secreted proteins that are expressed primarily in hematopoietic tissues and are potential serum markers for certain hematopoietic malignancies. hFR-gamma' is a variant of hFR-gamma with a two base deletion in its cDNA resulting in a truncated polypeptide. The gene encoding hFR-gamma' was isolated from a placental genomic library. The gene has five exons, four introns and a 5' flanking sequence which contains multiple putative regulatory elements. From RNase protection assay and RACE analysis, the major site of transcriptional initiation was identified at -56 nt. Systematically deleted fragments in the 5' region of the genomic DNA of FR-gamma' were ligated into the PGL3Basic plasmid and the reporter luciferase activity was assayed in cell lysates from transiently transfected NIH3T3 cells. From those results, putative positive and negative regulatory regions in the 5' flanking sequences were noted, and a TATA-less proximal promoter was located between -206 and -22 nt. Gel mobility shift and supershift analyses as well as mutagenesis experiments revealed that Sp1 and ets binding elements in the proximal promoter region confer transcriptional activity. From partial sequencing of genomic DNA, genomic Southern blots, RACE analysis and RNase protection assays, it appears that hFR-gamma shares the gene organization of hFR-gamma'. The results of the analysis of genomic DNA in spleen tissues from several individuals, were consistent with the interpretation that hFR-gamma and hFR-gamma' are encoded by a polymorphic gene.

In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type alpha and type beta have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn-->Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-alpha and FR-beta are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-alpha and FR-beta respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-beta the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-alpha and FR-beta.

The distribution of the glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) in a diffuse pattern vs. functional clusters associated with caveolae has been debated. The equivocal nature of direct localization studies is due to possible experimental artifacts such as cross-linking of the protein by the antibody probes prior to fixation and alternatively the use of a disruptive fixation method. Such studies have also been complicated by the use of cells that vastly overexpress FR. In this study a monovalent probe, i.e., a biotinylated folate affinity analogue was used to covalently label FR. Cells expressing moderate levels of FR, i.e., JAR epithelial cells expressing FR-alpha and recombinant CHO fibroblasts expressing FR-beta, were used. The affinity label and either caveolin or antigenic sites on FR were localized by electron microscopy using colloidal gold conjugated antibody probes post-embedding in the relatively permeable LR White resin. The method avoided both receptor cross-linking and early fixation steps and also enabled the use of transport permissive conditions while labeling FR at the cell surface. The results indicate that in steady-state FR is not significantly colocalized with caveolin. However, the receptor molecules occur predominantly in clusters, independent of cross-linking, providing a physical basis for the observed kinetics of receptor internalization and recycling during folate transport. Evidence is also presented to suggest that early mild fixation will disrupt the clustering of FR.

The homologous folate receptor (FR) types alpha and beta from both human and murine sources have opposite stereospecificities for reduced folate coenzymes and different affinities for a variety of (anti)folate compounds. The present study identifies the critical amino acid sequence divergence underlying functional differences between FR-alpha and FR-beta. Chimeric constructs of the cDNAs encoding human FR-alpha and FR-beta were expressed in human 293 fibroblasts. The resulting membrane associated proteins were characterized in terms of their ability to bind [3H]folic acid and their relative affinities for the (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate. Substitution of the amino-terminal portion (residues 1-92) in the mature FR-alpha polypeptide with the corresponding segment of FR-beta resulted in folate binding characteristics similar to FR-beta. Next, a series of chimeric constructs were generated, involving substitution of progressively shorter segments within residues 1-92 in FR-alpha with the corresponding peptides of FR-beta. In this fashion, it was determined that the alanine residue at position 49 in FR-alpha was critical for its functional divergence from FR-beta, since substitution at this position with Leu (the corresponding residue in FR-beta) resulted in the folate binding characteristics of FR-beta. Reciprocal substitution in FR-beta with peptide 1-92 of FR-alpha resulted in poor expression of a [3H]folic acid binding protein. By analysis of chimeric constructs, the poor [3H]folic acid binding of the FR-alpha(1-92)/beta(93-237) chimera could be attributed to interference of a short segment from FR-alpha in the vicinity of Ala 49 (peptide 39-59) with proper folding of the chimera. Conversion of the ligand binding properties of FR-beta to those of FR-alpha required the reciprocal mutation of Leu 49 to Ala, but in addition, substitution of one or more residues downstream of amino acid 92 of FR-beta with the corresponding residues in FR-alpha was essential. The homologous murine FR types alpha and beta, which are functionally analogous to the human receptor isoforms, also contain a similar Ala vs Leu substitution. These results indicate that steric/hydrophobic effects of the side chains of Leu vs Ala at position 49 will critically modulate the affinities and stereospecificities of FR isoforms for folate compounds. Furthermore, additional amino acid sequence divergence at one or more positions downstream of residue 92 in FR-alpha is also an essential determinant of the unique functional characteristics of this receptor isoform.

Two variant sublines of murine L1210 leukemia cells (L1210A and L1210JF) overexpress the cell surface folate receptor (FR). The membrane bound FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N5-methyltetrahydrofolate, (6S)-N5-formyltetrahydrofolate and methotrexate compared to the FR in L1210JF cells. Furthermore, receptor-mediated transport of [3H]-(6S)-N5-methyltetrahydrofolate was much more efficient in L1210A cells compared to L1210JF cells. When solubilized with Triton X-100, the ligand binding characteristics of FR from both sublines resembled those of the receptor associated with L1210 JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revealed a single species of FR in both cells, identical to murine FR-alpha. The FR in L1210JF cells was sensitive to phosphatidylinositol specific phospholipase C (PI-PLC) indicating the presence of a glycosyl-phosphatidylinositol (GPI) membrane anchor while the FR in L1210A cells was resistant to PI-PLC; however, the FR in L1210A cells was released from plasma membranes by nitrous acid, as expected for GPI and its PI-PLC resistant structural variants. Treatment of L1210A cell membranes with mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affinities of the membrane associated FR for reduced folates. When the cDNA for murine FR-alpha was expressed in parental L1210 cells the protein was PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expressed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [3H]ethanolamine, a component of the GPI membrane anchor; however, the labeled proteins were released by PI-PLC only in L1210JF cells. The above results preclude any peculiarity of the FR polypeptide in either L1210 subline as the basis for the observed differences in PI-PLC sensitivity and membrane-associated functions of FR. Partial deglycosylation of membrane associated FR from either cell with N-glycanase did not influence its ligand binding characteristics. The results of this study lead to the hypothesis that variant GPI structures may modulate the function of a protein by influencing its conformation/topography in the membrane. Such effects may be identified by their disappearance/reduction upon detergent solubilization or mild base treatment of the membrane.

Two methotrexate (MTX)-resistant human breast-cancer cell lines with impaired transport via the reduced folate carrier (RFC), one established in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231), were adapted to grow in medium containing 2 nM folic acid. This induced the expression of previously undetectable membrane folate receptors (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Polymerase chain reaction (PCR) quantitation revealed that MFR messenger-RNA levels of the isoform first described in human nasopharyngeal carcinoma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)-ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth-inhibitory effects of five different antifolates with varying affinities for MFR: N10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-dideazatetra-hydrofolic acid (DDATHF) > N-5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-methyl) -N-methyl-amino]-2-theonyl}-glutamic acid (ZD1694) >> MTX > edatrexate (EDX). Expression of MFR only slightly decreased the resistant phenotype for MTX, EDX, and ZD1694, suggesting that these drugs are not transported intracellularly to cytotoxic concentrations at these levels of MFR expression. On the other hand, both cell lines became from at least 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. These sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affinity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cells with the highest level of MFR expression (MTX(R)-ZR-75-1). These results illustrate that the efficacy of MFR in mediating antifolate transport and cytotoxicity depends on their affinity for the folate antagonist, their degree of expression, and the levels of competing folates.

The divergent carboxyl-terminal signal peptides for glycosylphosphatidylinositol (GPI) membrane anchor attachment in folate receptor (FR) types alpha and beta were characterized. All of the candidate amino acid residues for GPI modification were identified and tested by substituting individually and in combination with amino acids that cannot be modified by GPI. Thus the GPI modification in FR-alpha was decreased to 22% by mutation of Ser234 to Thr but unaltered by changing the other candidate, Gly235, to Met. However, the double mutant FR-alpha Ser234-Thr,Gly235-Met showed half of the GPI modification seen in FR-alpha Ser234-Thr. This result suggests that Ser234 is the preferred GPI modification site, while Gly235 is a minor, alternate GPI modification site. Similarly, in FR-beta, mutation of Asn230 to Gln decreased GPI modification to 32%, while mutation of the other candidate site, Gly237, to Met had no effect. However, mutation at both sites further reduced the GPI modification by a half. A five amino acid carboxyl-terminal deletion (FR-beta delta 5) caused no decrease in the extent of GPI modification. However, the same deletion in FR beta Asn230-Gln decreased the residual GPI modification by 66%. These results suggest that Asn230 is the preferred GPI modification site in FR-beta, while Gly235 offers a minor alternate modification site; consistent with this conclusion is the fact that modification at the downstream site is hindered by its proximity to the carboxyl terminus in FR-beta delta 5. Further, the suggestion that the hydrophobic portion of the GPI signal is a random sequence of neutral amino acids with overall moderate hydrophobicity was tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on differential levels of membrane-associated folate binding protein (mFBP) expression, murine L1210 leukemia, human KB epidermoid carcinoma, and human IGROV1 ovarian carcinoma cells maintained under low (physiological) folate conditions (2 nM folinic acid) were used as model systems to investigate the potential role of mFBP in antifolate transport. In addition, L1210 parental cells were compared to a subline, L1210A, expressing high levels of mFBP and defective reduced folate carrier. Antifolates for which KB-derived mFBP has high affinity (5, 10-dideazatetrahydrofolic acid [DDATHF] and homo-DDATHF [0.24 and 0.78 respectively relative to folic acid]) and low affinity (methotrexate [0.002]) were chosen for this study. Protection against DDATHF/homo-DDATHF induced cytotoxicity was achieved preferentially by folic acid compared to folinic acid in IGROV1 and L1210A cells. In IGROV1 cells, cytotoxicity IC50s were increased 18- and 5.5-fold for DDATHF and homo-DDATHF respectively by 20 nM folic acid. Moreover, greater protection was observed in L1210A cells, where IC50s were increased 354- and 80-fold for these same compounds by 20 nM folic acid. Similar protection was not observed in KB cells, suggesting that KB mFBP was not functional in DDATHF transport. Although mFBP expression may be an important determinant in the cytotoxicity of antifolates for certain tumor cells, our data demonstrate a lack of correlation between levels of mFBP and function of mFBP for DDATHF transport in the models studied.

Background: Despite significant differences in ligand binding between the two known isoforms of the human membrane folate receptor (FR), designated herein as FR-beta (placenta) and FR-alpha (placenta, KB cells), little is known about their tissue specificities, and there is no report on the relative expression of FR-beta in any tissue other than in placenta., Methods: The mRNA for each FR isoform in a wide variety of normal fetal and adult tissue explants, primary normal cell cultures, malignant tumor explants, and established tumor cell lines was estimated by a polymerase chain reaction assay. Total receptor levels were estimated by a [3H] folic acid binding assay., Results: Both the FR isoforms were expressed in fetal as well as adult tissues. Normal tissues generally expressed low to moderate amounts of FR-beta. FR-alpha alone was expressed in normal epithelial cells and was frequently strikingly elevated in a variety of carcinomas, with the exception of squamous cell carcinomas of the head and neck. In contrast, a variety of malignant tissues of nonepithelial origin generally expressed elevated levels of FR-beta alone. Established tumor cell lines expressed FR-alpha virtually alone and did not reflect FR expression patterns in vivo. KB cells and JEG-3 cells grown at low folate concentrations further up-regulated FR-alpha but not FR-beta., Conclusions: Although FR-beta is the more common isoform, FR-alpha and FR-beta are differentially regulated in normal tissues, carcinomas, nonepithelial malignancies, and immortalized cells or in response to changes in extracellular folate concentrations. The tissue specificity of FR isoforms and their elevation in malignant tissues may be a significant factor in FR-mediated folate uptake, in tissue responsiveness to promising novel antifolates, and in FR-related immunodiagnosis/immunotherapy.

The expression of a membrane-associated folate receptor (FR) was elevated in spleen samples from patients with chronic (CML) and acute (AML) myelogenous leukemias compared with normal spleen. Contrary to earlier reports, antibodies to a purified FR from placenta cross-reacted quantitatively with this protein in solution radioimmunoassays. Similar to FR-alpha (KB cells) and FR-beta (placenta), the protein was released from the membrane by phosphatidylinositol-specific phospholipase C, indicating a glycosylphosphatidylinositol (GPI) membrane anchor. Screening of a cDNA library from CML spleen with a heterologous murine FR cDNA and also amplification of FR cDNAs from spleen and bone marrow in CML, AML, chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL) by polymerase chain reaction (PCR) using degenerate oligonucleotides yielded cDNA clones representing FR-beta, a novel FR (type gamma), and an aberrant transcript of FR-gamma with a 2 base pair deletion resulting in a truncated 104-residue polypeptide; FR-alpha was not detected in these tissues. The cDNA for FR-gamma predicts a 243-residue polypeptide with an amino acid sequence homology of 71% and 79% with FR-alpha and FR-beta, respectively, a 23-residue amino-terminal signal peptide, and 3 potential sites for N-linked glycosylation. Transfection of COS-1 cells with the cDNA for FR-gamma resulted in low expression of a [3H]folic acid binding protein on the cell surface that was GPI-anchored. PCR analysis of total RNA from a number of normal and malignant tissues and cell lines indicated a limited tissue specificity of FR-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.

A variant line (CEM-7A) "overproducing" the reduced folate/MTX carrier system was isolated from human CCRF-CEM leukemia cells grown under selective conditions in medium containing 0.25 nM 5-formyl-THF as the sole folate source. This line exhibits a 95-fold increased Vmax for [3H]-MTX influx as compared to parental cells. The values for [3H]-MTX influx Km, efflux t1/2 and structural specificity for other (anti)folate compounds were unchanged. The amount of carrier protein, estimated by NHS-[3H]-MTX affinity labeling, was approximately 30-fold higher in CEM-7A cells than in parental cells. Influx of [3H]-MTX in CEM-7A cells was found to be down-regulated 6-7-fold after preincubation of cells with adenosine, 5-formyl-THF or 5-methyl-THF, but could be prevented exclusively by inhibitors of dihydrofolate reductase. The underlying mechanism(s) of these effects have not as yet been elucidated. A radioiodinated photoaffinity analog of MTX was used to prove the molecular events in carrier-mediated MTX uptake in parental CCRF-CEM cells, CEM-7A cells, and a line exhibiting a MTX-transport defect (CEM-MTX). Specific labeling of an 80-85 kDa membrane protein was observed in parental cells, but not in CEM/MTX cells. Uptake of photoprobe and levels of the 80-85 kDa membrane protein were significantly increased in CEM-7A cells. Due to extensive glycosylation the MW of the carrier protein in human cells seems to be substantially higher than that of its counterpart in murine L1210 leukemia cells (46-48 kDa). Pulse-labeling experiments at 37 degrees C demonstrated that in CEM-7A cells photoprobe uptake proceeds via a specific pathway. The 80-85 kDa membrane protein is involved in the initial binding and translocation of photoprobe, after which a 38 kDa cytosolic protein is responsible for further intracellular distribution. At this time, the combination of photoaffinity labeling techniques and the availability of variant cell lines overexpressing the reduced folate/MTX carrier protein has provided new insights into the MTX transport process in human leukemia cell lines. In the near future this approach should also allow a further elucidation of the regulatory aspects of carrier function.

A preparation of folate binding protein purified from human placental membranes in the presence of a variety of protease inhibitors followed by deglycosylation with N-glycanase gave a sharp band at Mr approximately 28,000 following SDS-polyacrylamide gel electrophoresis. The deglycosylated protein bound [3H]folic acid as tightly as the native protein. Peptides obtained following digestion of the purified protein with staphylococcal V8 protease and HPLC purification were sequenced. Polyclonal antibodies against the protein preparation were affinity purified and used to screen a placental cDNA expression library. A full-length cDNA for a placental folate binding protein was thus obtained and the corresponding protein sequence deduced. This result, taken together with the peptide sequence data, indicates the expression of at least two homologous folate binding proteins in placenta, one of which appears to be identical with the folate binding protein from human milk and nasopharyngeal epidermoid carcinoma (KB) cells; the cDNA sequence obtained corresponds to the other protein. The deduced protein sequence is characterized by a putative 16-residue amino-terminal signal peptide that is cleaved, resulting in a 239-residue polypeptide. The mature protein exhibits two potential sites for N-linked glycosylation at Asn-99 and Asn-179, eight potential intramolecular disulfide bonds, and a stretch of hydrophobic residues at the carboxyl terminus that could form a transmembrane domain. The protein bears a 68% sequence homology with the KB cell folate binding protein and may represent a fetal folate transport protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The transport systems for folate coenzymes and antifolate compounds into various types of normal and neoplastic cells display considerable diversity in their pharmacokinetics and also in terms of the apparent molecular weights of the proteins involved. Further, several uptake routes may exist in a given cell type. A variety of neoplastic tissues have been reported to rely upon a single major transport system that has a relatively high affinity for the reduced form of folate compounds and for antifolates such as methotrexate. Using a photoaffinity analogue of methotrexate, we have identified the involvement of a 48 kDa membrane protein and a 38 kDa cytosolic or peripheral membrane protein in the transport of this compound into murine L1210 leukemia cells. Such an uptake is absent in mutant L1210 cells that are defective in methotrexate transport. We propose a model for the uptake of reduced folate coenzymes in L1210 cells in which the compound is initially transported across the cell membrane by the 48 kDa protein and delivered on the cytoplasmic surface to the 38 kDa protein; the 38 kDa protein then carries the folate compound to a specific enzyme of folate metabolism. Antibodies to the membrane folate binding protein from human placenta cross-react with the 48 kDa protein in L1210 cell membranes indicating an immunological relationship between these two proteins. Comparison of the amino acid sequences of peptides of the placental receptor obtained by digestion with S. aureus V8 protease indicate the presence of two homologous forms of the folate binding protein in placenta; one of these forms appears to have an identical sequence to the soluble and membrane associated folate binding proteins in human epidermoid carcinoma (KB) cells, which in turn share the primary structure of the soluble and membrane associated folate binders in human milk in the regions that have been sequenced. These results indicate that the folate coenzyme transport proteins in various tissues may be structurally related and in several instances may even be identical. In the latter cases the observed differences in apparent molecular weights may be due to differences in glycosylation and/or proteolysis. In support of this view is our observation that the deglycosylated and/or partially proteolyzed placental receptor retains the ability to bind folate.

A radioiodinated photoaffinity analogue of methotrexate, N alpha-(4-amino-4-deoxy-10-methyl-pteroyl)-N epsilon-(4-azidosalicylyl)-L- lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., & Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degrees C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degrees C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)