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K N Chung

Publications and source records attributed to K N Chung.

11 recordsLinked to original sources
PubMed1998

Role of individual N-linked glycosylation sites in the function and intracellular transport of the human alpha folate receptor.

Glycosylation is a structural feature of all three isoforms of the human folate receptor. We have used site-directed mutagenesis to study the role of individual glycosylation sites in the assembly and function of the a isoform of the human folate receptor (alpha(h)FR). Three potential N-linked glycosylation sites in the alpha(h)FR sequence were disrupted by conservative mutation of the S or T residues in the consensus sequence (N-X-S/T) to A or V, respectively. Constructs with the single mutations S(71)-->A (alpha(h)FR(-1)), T(163)-->V (alpha(h)FR(-2)), and S(203)-->A (alpha(h)FR(-3)); the double mutation S(71)--> A/S(203)-->A (alpha(h)FR(-1-3)); and the triple mutation S(71)--> A/S(203)--> A/T(163)--> V (alpha(h)FR(-1-2-3)) were stably transfected into Chinese hamster ovary (CHO) cells. The proteins produced in CHO cells by the mutated cDNAs have apparent molecular weights that are reduced relative to the wild type and are consistent with the loss of carbohydrate residues. The triple mutant, which lacks all three consensus glycosylation sites, yields protein that comigrates with the enzymatically deglycosylated native protein. Determinations of the K(D) for folic acid by Scatchard analyses of the glycosylation mutants indicate that folic acid binding affinity is not significantly affected in the single mutants alpha(h)FR(-1) and alpha(h)FR(-2). However, in the single mutant, alpha(h)FR(-3), and the double mutant, alpha(h)FR(-1-3), folic acid binding affinity is respectively 2.7- and 3.5-fold lower than that in wild type. Deglycosylation by mutation of all three consensus sites (alpha(h)FR(-1-2-3) eliminates both folic acid binding and cell surface expression. In contrast, enzymatic deglycosylation of purified wild-type alpha(h)FR with endoglycosidase F does not significantly affect folate binding affinity. Thus, while carbohydrate residues are not essential for the folate binding activity of the mature folate receptor, at least one of the three core glycosylated residues is necessary for the synthesis of alpha(h)FR in its active conformation.

Animals↗
PubMed1997

Tissue-specific promoters of the alpha human folate receptor gene yield transcripts with divergent 5' leader sequences and different translational efficiencies.

The alpha human folate receptor (alphahFR), or KB cell folate receptor, gene contains two major promoters that produce transcripts, KB1 and KB4, varying only in the length and sequence of their 5' untranslated regions (UTRs). Using RNase protection assays specific for each isoform, we show that the level of expression of these two transcripts is tissue-specific, indicating that promoter usage is regulated, not constitutive. RNA stabilities and translational efficiencies of the KB1 and KB4 transcripts were compared to determine the functional significance of the different 5' UTRs. Analyses of RNA turnover in vivo with actinomycin D to block new transcription and in vitro with a cytoplasmic extract indicate no discernible differences in the stabilities of the two transcripts. However, the KB4 transcript is 2-3-fold more efficiently translated in wheat germ extracts in vitro and transfected CHO cells in vivo. Also, high ionic strength, which favours the formation of RNA secondary structure, differentially affects the translational efficiencies of the two transcripts. Translation of the longer KB1 mRNA is 2-5-fold more inhibited by hypertonic conditions than translation of the KB4 mRNA. Because the 5' UTR of KB1 is approximately four times longer than the 5' UTR of KB4, 149 bp (75%) of the KB1 5' UTR were deleted to determine whether the long leader sequence inhibited translation. The resulting derivative, dKB1, has a 5' UTR similar in length, but not sequence, to the 5' UTR of KB4. dKB1 is translated at a level approaching that of KB4 in wheat germ extracts, indicating that the upstream portion of the 5' leader sequence contributes to the relative translational inefficiency of KB1. Hence, one consequence of tissue-specific promoter usage is the production of alphahFR transcripts with different 5' non-coding regions that affect translational efficiency.

Animals↗
PubMed1997

The divergent 5' termini of the alpha human folate receptor (hFR) mRNAs originate from two tissue-specific promoters and alternative splicing: characterization of the alpha hFR gene structure.

The human KB cell or alpha folate receptor (alpha hFR) is a membrane glycoprotein of 42 kDa that participates in the internalization of folates and antifolates. Seven independent alpha hFR cDNA isoforms have been reported that contain unique 5' termini but share a common open reading frame (ORF). To investigate the molecular basis of these heterogeneous 5' sequences, we determined the sequence of the alpha hFR gene from two clones isolated from a human lymphocyte lambda DASH genomic library. The gene is composed of seven exons that span 6.8 kb. The ORF is encoded by exons 4 through 7 while the reported 5' termini of the cDNA isoforms (including two novel cDNAs designated KB2 and KB4) are encoded by exons 1 through 4. Using RNase protection assays, we demonstrate that transcripts corresponding to the KB1 and KB4 cDNAs originate from promoters upstream from exon 1 and exon 4, designated P1 and P4, respectively, and that these mRNA isoforms are the most abundant transcripts expressed in KB cells and selected normal tissues (including kidney, lung, and cerebellum). We observed a heterogeneous start site within exon 1 from the P1 promoter while transcripts from the P4 promoter originate from a single site. In addition, we detected tissue specificity for the P1 and P4 promoter utilization. Transcripts originating from the P1 promoter are the most abundant transcripts expressed by human cerebellum and kidney. In contrast, transcripts from the P4 promoter are the most abundant transcripts expressed by human KB cells and lung. Total RNA from KB cells also protects a 66 bp fragment of an exon 3 riboprobe that is consistent with an alternatively spliced transcript. To examine the functional activity of the predicted P1 and P4 promoters, alpha hFR promoter-CAT chimeric plasmids were constructed using sequences flanking exon 1 and exon 4. We observed a 7.5- and 10-fold increase in CAT activity in HeLa cells transiently transfected with the P1 and P4 promoter constructs, respectively. These data demonstrate that a single gene encodes the divergent 5' termini of the alpha hFR cDNAs and that the alpha hFR transcripts are transcribed from two promoters that are activated in a tissue-specific manner.

Alternative Splicing↗
PubMed1996

Identification and characterization of a radiation-inducible glycosylated human early-response gene.

A radiation-inducible immediate-early gene, IEX-1, was identified and characterized in human squamous carcinoma cells. Sequence analysis revealed 156-amino acid nucleotides, encoding a protein of Mr 20,000. The protein is glycosylated (Mr approximately 27,000) in the presence of microsomal membranes. Northern analysis reveals a 1.2-kb transcript. Treatment with cycloheximide was associated with superinduction of this transcript suggesting that it is an immediate-early gene. The abundance of IEX-1 mRNA increased rapidly after exposure of the cells to ionizing radiation (2-10 Gy), reaching a maximum by 15 min and returning subsequently to basal levels by 4 h. Expression of IEX-1 was also induced significantly by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein phosphatase inhibitor okadaic acid, and tumor necrosis factor-alpha, whereas treatment of cells with UV light and H2O2 had little effect on IEX-1 expression. Cells depleted of PKC by prolonged incubation with TPA showed no attenuated IEX-1 response to tumor necrosis factor-alpha. This is the first report of IEX-1, a radiation-inducible glycosylated human protein, whose expression can be mediated through multisignal transduction pathways.

Amino Acid Sequence↗
PubMed1995

Rapid turnover and impaired cell-surface expression of the human folate receptor in mouse L(tk-) fibroblasts, a cell line defective in glycosylphosphatidylinositol tail synthesis.

The human folate receptor (hFR) is a plasma membrane protein that is anchored to the membrane via a glycosylphosphatidylinositol (GPI) tail in some cell types. The KB hFR cDNA sequence predicts a hydrophobic, alpha-helical 31-residue carboxyl terminus that is thought to be the signal for cleavage and attachment of the GPI tail. Alternatively, this region may serve as a transmembrane domain if GPI attachment is not efficient. In this study, we investigated the latter possibility by expressing the hFR in L(tk-) cells, cells that are unable to synthesize GPI tails for attachment to membrane proteins. We also transfected the same hFR cDNA into Chinese hamster ovary (CHO) cells, cells that can anchor proteins by either a GPI tail or a transmembrane domain. Neither parental cell line expresses detectable levels of folate receptor as determined by folic acid binding assays, Western analysis, or Northern analysis. In L(tk-) cells, we found that the recombinant hFR is not expressed on the cell surface, but is rapidly degraded (t1/2 < or = 4 h). Most (> 95%) of the recombinant hFR remains Endo H sensitive, suggesting retention in the endoplasmic reticulum. In contrast, transfected CHO cells express functional hFR protein at the cell surface, the half-life of the hFR is long (t1/2 > or = 24 h), and the Endo H glycosylation pattern of the recombinant hFR is consistent with normal processing through the Golgi apparatus. Therefore, in the absence of a GPI tail, the hFR is not sorted to the cell surface and the incompletely processed hFR protein is unstable.

Animals↗
PubMed1994

Site-directed mutagenesis of tryptophan residues to conserved hydrophobic residues inhibits the processing of human KB cell folate receptor.

We are interested in identifying the ligand binding site of the human folate receptor (hFR). Previous reports have suggested a role of tryptophan(W) residues in ligand binding. We used site-directed mutagenesis to change the conserved W residues in positions 86, 116, 142, 143, 156, 160, and 193 of the hFR to either leucine(L) or phenylalanine(F) to examine the role of these W residues in hFR function. Although all W to L changes except W86L produced unstable proteins, W to F changes were tolerated. Based on total folate binding and transport studies, Chinese hamster ovary (CHO) cells transfected with W86L, W116F, and W143F expressed high levels of functional hFR, equivalent to cells transfected with wt hFR. CHO cells transfected with W142F, W156F, W160F, and W193F expressed low or undetectable levels of functional hFR although mRNA was present. Of these four mutants, only W142F expressed easily detectable immunoprecipitable protein but it was not fully glycosylated. Since glycosylation may affect the ability of hFR to bind folate, we expressed W142F in Xenopus oocytes which glycosylate the mutant and wild type proteins to the same apparent extent. In oocytes, the stoichiometry of folate binding was identical between the fully processed mutant protein and the wild type hFR. These results indicate that in CHO cells three of the seven W mutations (W86L, W116F, and W143F) function normally, whereas four of the seven W mutations (W142F, W156F, W160F, and W193F) produce unstable or abnormally processed protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.

Animals↗
PubMed1992

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.

Biological Transport↗
PubMed1989

Molecular sorting in the secretory pathway.

Proteins can be secreted from animal cells by either a constitutive or a regulated pathway; those destined for regulated secretion are actively sorted into dense-core secretory granules. Although sorting is generally assumed to be accomplished by specific carriers, the nature of these carriers remains elusive. In this study, peptide hormones were used as affinity ligands to purify a set of 25-kilodalton proteins from canine pancreatic tissue. Their ligand specificities and patterns of expression have the characteristics of sorting carriers.

Animals↗
PubMed1984

Swainsonine and castanospermine blockade of mannose glycoprotein uptake by macrophages. Apparent inhibition of receptor-mediated endocytosis by endogenous ligands.

Receptor-mediated uptake of mannose-terminated glycoproteins by macrophages is blocked by treating the cells with swainsonine, an inhibitor of alpha-mannosidase II, and by castanospermine, an inhibitor of the endoplasmic reticulum processing enzyme alpha-glucosidase. Both inhibitors are known to cause accumulation of unprocessed oligosaccharide chains terminating in mannose. Inhibition of ligand uptake by the drugs was time- and dose-dependent. Swainsonine produced a maximal effect after 2 h; castanospermine required 5-6 h. Following swainsonine treatment, complete recovery of mannose receptor activity required 24 h and was blocked by cycloheximide suggesting that new receptor synthesis was necessary. Tunicamycin, an inhibitor of oligosaccharide assembly, had no effect on uptake of mannosylated ligands, but tunicamycin pretreatment reduced the sensitivity to swainsonine. These effects of swainsonine and castanospermine appear to be specific, other macrophage pinocytosis receptors (e.g. mannose phosphate) or phagocytosis of yeast particles were unaffected. Moreover, swainsonine had no effect on the fibroblast mannose phosphate receptor. The ability of macrophages to process newly synthesized oligosaccharides was blocked following treatment with swainsonine. Normal processing was fully recovered 24 h after removal of the drug. Mannosidase II was partially inactivated by swainsonine treatment and only a portion was recovered after 24 h. Treatment of macrophages with swainsonine also resulted in an increase in net lysosomal enzyme secretion. Inhibition of mannose-specific receptor-mediated endocytosis in macrophages by swainsonine and castanospermine appears to be due to the formation of mannose-terminated membrane glycoproteins which engage the mannose receptor thereby preventing function. These results suggest a novel mechanism for regulation of receptor-mediated endocytosis.

Alkaloids↗