Down-expression of miR-373 predicts poor prognosis of glioma and could be a potential therapeutic target.

OBJECTIVE: MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including glioma. The main objective of this work was to investigate the expression level of miR-373 and its clinical significance in glioma. PATIENTS AND METHODS: The expression levels of miR-373 in glioma tissues and non-neoplastic brain tissues were measured by the qRT-PCR assay. Patients were divided into two groups based on the median miR-373 expression. The probability of differences in overall and progression-free survival as a function of time was ascertained by use of the Kaplan-Meier method. Cox regression analysis of factors potentially associated with survival was conducted to identify independent factors. RESULTS: In clinical gastric cancer samples, we found that miR-373 expression was significantly down-regulated in glioma tissues compared with non-neoplastic brain tissues (p<0.01). Reduced expression of miR-373 was associated with serum WHO grade (p=0.015) and KPS score (p=0.001). Kaplan-Meier analysis indicated that patients with low level of miR-373 expression had poorer overall survival (OS) and progression-free survival (PFS). Multivariate survival analysis verified that miR-373 expression level was an independent predictor of both OS and PFS for glioma patients. CONCLUSIONS: Our study showed miR-373 was associated to progression in glioma, and suggested it as a potential predictive factor for the prognosis of glioma.

Identification of two isoforms of the Cak receptor kinase that are coexpressed in breast tumor cell lines.

The Cak receptor kinase is a member of a novel family of receptors that are characterized by the unique structure of the ectodomains. We have identified a new isoform of Cak that differs from the original isolate by the deletion of 37 amino acids in the cytoplasmic juxtamembrane sequence. Analysis of the genomic sequence suggests that the two isoforms arise by exon skipping. The isoform-specific insert contains the motif NPXY, which was previously shown to be involved in diverse signaling function in a number of receptors. By RNase protection analyses, we found that the long isoform, Cak I is expressed at three- to sevenfold the abundance of the short isoform (Cak II). By Western blotting, Cak I receptor was found to be expressed in mouse embryos and in adult brain. Cak II protein was not detected in mouse embryos or adult tissues, but is abundantly expressed in some breast tumor cell lines. The expression profile of Cak suggests that its primary function is likely to be in developmental regulation. The coexpression of the Cak isoforms in some epithelial cell lines suggests that heterodimer formation may be a key feature in the function of the receptor.

The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3.

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

The cDNA sequence and primary structure of the chicken transferrin receptor.

Recombinant cDNA clones encoding the chicken transferrin receptor (cTR) have been isolated and sequenced. Comparison of the deduced primary structure of cTR with those of the human transferrin receptor (hTR) and mouse transferrin receptor (mTR) shows that their size, hydropathy profile, location of sites for posttranslational modifications, and domain organization are highly similar. The cytoplasmic domain of cTR contains the motif Tyr-Xaa-Arg-Phe (YXRF) that is the recognition signal for high-efficiency endocytosis of hTR. The cTR has several highly conserved regions within its extracellular domain, including those flanking the putative N-glycosylation sites. Overall, however, the extracellular domain of cTR is only 53% identical to the extracellular domains of hTR and mTR. The cTR also lacks three of the six Cys residues found in the extracellular domains of the mammalian TRs. These differences can account for functional and structural properties that distinguish cTR and mammalian TRs.

The trk proto-oncogene encodes a receptor for nerve growth factor.

Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.

Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis.

Using detailed functional studies on 24 human transferrin receptor mutants, we identified YXRF as the internalization sequence. Provided that at least 7 residues separate this tetrapeptide from the transmembrane region, changing the tetrapeptide position within the TR cytoplasmic domain does not reduce internalization activity. Thus, any conformational determinant for internalization must be localized to the YXRF sequence. Twenty-eight tetrapeptide analogs of YXRF, found by an unbiased search of all known three-dimensional protein structures, significantly favored tight turns similar to a type I turn. Of the ten tetrapeptides most closely related to YXRF, eight were surface exposed and had tight-turn conformations, as were four of five tetrapeptides with sequences related to the low density lipoprotein receptor internalization motif, NPXY. The internalization sequences of both receptors contain aromatic residues with intervening hydrogen-bonding residues. Thus, two distinct internalization sequences favor a common structural chemistry and implicate an exposed tight turn as the recognition motif for high efficiency endocytosis.

Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake.

The human transferrin receptor is post-translationally modified by the addition of a fatty acyl moiety. In earlier studies, transient expression in Cos cells of human transferrin receptors in which Cys62 or Cys67 was altered to serine provided evidence that Cys62 is the major acylation site of the receptor (Jing, S., and Trowbridge, I. S. (1987) EMBO J. 6, 327-331). To determine whether acylation of the receptor is required for high efficiency endocytosis and iron uptake, wild type and mutant human transferrin receptors have been stably expressed in chick embryo fibroblasts using a helper-independent retroviral vector. In marked contrast to Cos cells, both Cys62 and Cys67 of the wild type human transferrin receptor were acylated in chick embryo fibroblasts. Moreover, their modification to serine did not abolish palmitate labeling, implying that one or both of these serine residues could serve as alternative lipid attachment sites in these cells. The relative labeling of mutant receptors with palmitate and the susceptibility of their lipid moieties to cleavage by hydroxylamine were consistent with Ser67 but not Ser62 serving as a lipid attachment site. Consequently, to obtain human transferrin receptors lacking covalently bound lipid in the chick embryo fibroblasts, it was necessary to alter Cys62 and Cys67 to alanine. Functional studies indicated that these non-acylated mutant receptors were internalized efficiently and mediated iron uptake from human transferrin at a similar rate to that of wild type receptors. We conclude, therefore, that acylation of the human transferrin receptor is not essential for endocytosis and recycling.

Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization.

Wild-type and mutant human transferrin receptors have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. The internalization of mutant human transferrin receptors, in which all but four of the 61 amino acids of the cytoplasmic domain had been deleted, was greatly impaired. However, when expressed at high levels, such "tailless" mutant receptors could provide chicken embryo fibroblasts with sufficient iron from diferric human transferrin to support a normal rate of growth. As the rate of recycling of the mutant receptors was not significantly different from wild-type receptors, an estimate of relative internalization rates could be obtained from the distribution of receptors inside the cell and on the cell surface under steady-state conditions. This analysis and the results of iron uptake studies both indicate that the efficiency of internalization of tailless mutant receptors is approximately 10% that of wild-type receptors. Further studies of a series of mutant receptors with different regions of the cytoplasmic domain deleted suggested that residues within a 10-amino acid region (amino acids 19-28) of the human transferrin receptor cytoplasmic domain are required for efficient endocytosis. Insertion of this region into the cytoplasmic domain of the tailless mutant receptors restored high efficiency endocytosis. The only tyrosine residue (Tyr 20) in the cytoplasmic domain of the human transferrin receptor is found within this 10-amino acid region. A mutant receptor containing glycine instead of tyrosine at position 20 was estimated to be approximately 20% as active as the wild-type receptor. We conclude that the cytoplasmic domain of the transferrin receptor contains a specific signal sequence located within amino acid residues 19-28 that determines high efficiency endocytosis. Further, Tyr 20 is an important element of that sequence.

Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.

Structural studies of the human transferrin receptor have shown that the molecule is a disulfide-bonded dimer consisting of two identical subunits (Mr = 95,000) which are post-translationally modified by the addition of a fatty acyl moiety. Oligonucleotide site-directed mutagenesis has been used to obtain mutant molecules in which each of the four cysteines, residues 62, 67, 89 and 98, clustered within or adjacent to the membrane-spanning region were modified to serine. By first preparing mutants with only one of these cysteine residues modified to serine and then obtaining additional mutants in which different combinations of two cysteine residues were modified, we have shown that both cysteine 89 and cysteine 98, which are located in the extracellular domain of the receptor, are involved in intermolecular disulfide bonds. Further, we have identified cysteine 62 as the major site of acylation. Each of the mutant molecules is synthesized and transported to the cell surface when the modified human transferrin receptor cDNAs are transiently expressed in simian Cos cells. It should therefore now be possible to design experiments to determine whether these modified receptors bind transferrin normally and mediate iron uptake.