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1. The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues and multiple repeat sequences. The precursor of the laminin receptor is apparently smaller than the 67-kilodalton protein isolated from tissue. The apparent molecular weight on SDS-polyacrylamide gels of the rabbit reticulocyte cell-free translation product of selectively hybridized laminin receptor mRNA is 37,000. Antisera to three different domains of the cDNA-predicted receptor were used to study the relationship between the 37- and 67-kilodalton polypeptides. Antisera to cDNA-deduced synthetic peptides of the receptor immunoprecipitated a 37-kilodalton band both from cell-free translation products and from pulse-labeled cell extracts. On immunoblots of cell extracts, one antisynthetic peptide antiserum recognized only the 67-kilodalton receptor, while another antiserum identified both 37- and 67-kilodalton polypeptides, suggesting a precursor-product relationship between the two polypeptides. PMID: [2531008]
3. Based upon positive immunologic comparisons, protein and cDNA sequencing, and in vitro and in vivo immunogenicity studies we propose that carcinomas, sarcomas and lymphomas/leukemias of rodents and humans share 37 kDa Onco-Fetal Antigen [OFA] as a T and B-lymphocyte stimulating, universal tumor specific transplantation antigen [UTSTA]. In the past four years, biochemical studies from several laboratories studying laminin receptor protein and immunological studies of OFA from our laboratories independently converged. OFA protein and immature or precursor Laminin Receptor Protein [iLRP] are > 99% identical proteins based on amino acid and cDNA sequencing and immunobiology studies summarized here. Acquired expression of 37 kDa OFA/iLRP enables malignant tumor cells to penetrate laminin tissue and vessel barriers. 37 kDa OFA/iLRP activates precursor thymic anti-OFA/iLRP specific cytotoxic T cell which kill emerging pretumor cells. Our reported findings also demonstrate that OFA/iLRP can function to induce specific immunoregulatory CD8 T-suppressor cells secreting IL-10 which impair effector T-cell killing of emerging tumor cells non-specifically and thereby facilitate tumor progression. Potential applications of OFA/iLRP detection in early cancer formation, for monitoring patient T cell subclass responses to OFA/iLRP as a predictor of tumor progression, and the use of OFA/iLRP peptides for specific anti-tumor immunotherapy are presented. PMID: [10697612]
4. This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development. PMID: [16141072]
5. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]
6. We isolated a monoclonal antibody M108, which recognized 40 kDa protein (p40) in the cytoplasm, the perinuclear region in interphase and the perichromosomal region during mitosis. As reported previously, it was revealed from the immunofluorescent observation and the biochemical analyses that the nuclear p40 was associated both with the nuclear envelope and the chromatin DNA in interphase nuclei. In this report, we isolated the p40 from cytoplasmic particles, and identified it by extensive microsequencing as LBP/p40, which was considered to be a precursor of laminin binding protein p67 (LBP/p67). Epitope-tagged LBP/p40 was expressed in cultured cells, and the protein was localized in the nucleus as well as in the cytoplasm. Further analysis showed that the nuclear LBP/p40 was tightly associated with the nuclear structures. PMID: [8954992]
7. BL6 murine melanoma cells contain approximately 110,000 cell surface binding sites for the basement membrane glycoprotein laminin. Treatment of isolated melanoma cell plasma membranes with detergent yields a single class of laminin receptor. The receptor was purified 900 fold by laminin affinity chromatography. The isolated receptor has a Mr of 67,000 and binds laminin with high affinity: kd = 2 nm. The binding affinity of the isolated receptor was similar to that of the plasma membranes or the whole cells. Such a laminin receptor, isolated here for the first time, could facilitate the interaction of metastasizing tumor cells with the basement membrane. PMID: [6301485]
8. Skeletal muscle myofibers are each ensheathed by a continuous basal lamina consisting predominantly of type IV collagen, laminin and heparan sulfate proteoglycan. In order to identify laminin-binding components in the muscle cell surface, plasma membranes from mouse thigh muscle and from rat L6 myoblasts were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred samples with I-labelled laminin revealed a prominent band of approximate mol. wt. 68 000. A protein of this mol. wt. was isolated by affinity chromatography of muscle cell plasma membranes on laminin-Sepharose. The hydrophobic protein has an apparent mol. wt. of 68 000 and has a high content of serine, glycine and acidic amino acids. After detergent solubilization the purified protein binds to laminin-coated Sepharose beads at a higher rate than to beads coated with either fibronectin or collagen types I and IV. The interaction of the protein, called LB 68, with laminin was also studied after incorporation into synthetic lecithin vesicles. While detergent-solubilized LB 68 bound to I-labeled laminin only at lower than physiological ionic strength, liposome-incorporated LB 68 bound to laminin in the absence of detergents under physiological conditions. We propose that this protein is involved in the interaction of myoblasts with laminin substrates and thus may participate in the anchorage of the basal lamina in the plasmalemma of myotubes. PMID: [16453457]
9. We used affinity chromatography to isolate a specific laminin-binding protein from murine fibrosarcoma cells. These cells bind exogenous laminin to their surface with high affinity (Kd = 2 X 10(-9)M for laminin) with approximately 5 X 10(4) sites per cell. Laminin affinity chromatography of [35S]methionine-labeled cell extracts produced two distinct proteins. One was identified as Type IV (basement membrane) collagen based on its migration pattern on SDS gels and bacterial collagenase sensitivity. The other protein, which migrates as a single band or closely spaced doublet on reduced SDS gels, has a reduced molecular weight of 69,000. Using a nitrocellulose filter disk assay, we found that the latter protein specifically bound 125I-laminin with the same high affinity (Kd = 2 X 10(-9)M for laminin) as did intact fibrosarcoma cells. By iodinating intact cells, we demonstrated that this laminin-binding protein is on the cell surface. We conclude that this protein with reduced molecular weight of 69,000 is a subunit or component of a larger cell surface receptor protein for laminin in this fibrosarcoma model. This laminin receptor may mediate the interaction of the cell with its extracellular matrix. PMID: [6302102]
10. A 33-kDa polypeptide (termed p40), which shares an antigenic determinant with a laminin receptor and is under translational control, is believed to serve as a precursor to the receptor and to be related to the neoplastic state. The present study of subcellular localization of this protein shows it to be a cytoplasmic component not associated with the plasma membrane. Most of the cellular p40 was found to be associated with polyribosomes as well as with 40S to 60S cytoplasmic particles. Conditions that lead to polysome disruption also caused release of the polysomal form of p40 as smaller particles, and polysome reconstitution was accompanied by uptake of p40 into these structures. Because of the large abundance of this protein in the cells (six to eight copies per ribosome), it is unlikely that it represents a factor that associates with the 40S preinitiation complex. The p40-containing particles appear to represent a newly discovered structure involved in the process of polysome formation. PMID: [1374897]
11. Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells. PMID: [11689427]
12. Midkine (MK) is a heparin binding multifunctional protein that promotes cell survival and cell migration. MK was found to bind to 37-kDa laminin binding protein precursor (LBP), a precursor of 67-kDa laminin receptor, with K(d) of 1.1 nM between MK and LBP-glutathione-S-transferase fusion protein. The binding was inhibited by laminin, anti-LBP, amyloid beta-peptide, and heparin; the latter two are known to bind to MK. In CMT-93 mouse rectal carcinoma cells, LBP was mostly located in the cytoplasm as revealed by immunostaining with anti-LBP antibody. That a portion of LBP or 67-kDa laminin receptor was located at the surface of these cells was verified by inhibition of cell attachment to laminin-coated dishes by anti-LBP antibody. When MK was added to culture medium of these cells, a part of LBP migrated to the nucleus. The movement occurred concomitantly with nuclear transport of biotin-labeled MK. These findings suggested that the binding of MK to LBP caused nuclear translocation of the molecular complex. PMID: [11597123]
13. The 37 kDa/67 kDa laminin receptor LRP/LR acts as a receptor for both PrPc and PrPSc at the cell surface. Here, we further analyzed the subcellular localization of fluorescent labeled prion protein (PrP) and laminin receptor (LRP/LR) molecules. We show that EGFP-PrP is localized at the cell surface and in a perinuclear compartment, respectively. In contrast, a DsRed-DeltaSP-PrP mutant lacking the signal peptide is almost exclusively found in the nucleus but does not colocalize with heterochromatin. Interestingly, LRP-DsRed efficiently colocalizes with EGFP-PrP in the perinuclear compartment and LRP-ECFP partly colocalizes with DsRed-DeltaSP-PrP in the nucleus, respectively. We conclude that the interactions of PrP and LRP/LR are not restricted to the cell surface but occur also in intracellular compartments suggesting a putative role of LRP/LR in the trafficking of PrP molecules. PMID: [18339329]
14. Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 microg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at -80 degrees C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies. PMID: [19367708]
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