Identification and Characterization of Two Distinct Ligand Binding Regions of Cubilin

Yammani, Raghunatha R.; Seetharam, Shakuntla; Seetharam, Bellur

doi:10.1074/jbc.m106419200

Cited by 43 publications

(54 citation statements)

References 29 publications

(20 reference statements)

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“…Also, a number of the type II transmembrane serine proteases contain CUB domains thought to mediate enzyme-substrate interactions (Hooper et al, 2001). In addition, CUB domains of cubilin mediate binding to both the intrinsic factor-cobalamin as well as albumin (Yammani et al, 2001). As SIMA135/CDCP1 is heavily glycosylated within its extracellular domain, it is probable that ligand binding will be, at least partially, dependent on carbohydrate moieties as has been demonstrated for various isoforms of the cell surface glycoprotein CD44 (Bajorath, 2000).…”

Section: Discussionmentioning

confidence: 99%

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

et al. 2003

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We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M + HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M + HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/ CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M + HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/ CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

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Section: Discussionmentioning

confidence: 99%

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

et al. 2003

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“…There are several arguments, however, to suggest that megalin plays a role in cubilin function (30 -32). Cubilin and megalin are coexpressed in cells and co-localize (8,31), and both proteins interact in vitro (12,30,31). Moreover, knocking out the megalin gene or reducing megalin expression decreases cubilin expression and activity (30,32).…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in the cubilin gene have been identified in a group of patients who have Imerslund-Gräsbeck's syndrome (or megaloblastic anemia type 1) (6,7), a rare genetic disorder characterized by defective intestinal absorption of vitamin B 12 and proteinuria. The latter symptom is explained by the role of cubilin in the uptake of filtered plasma proteins including the vitamin D binding protein, albumin, hemoglobin, transferrin, Ig light chains, Clara cell secretory protein, and apolipoprotein A1.…”

mentioning

confidence: 99%

“…The protein contains a cleavable signal sequence that allows the polypeptide chain to enter the endoplasmic reticulum (ER), but it does not contain a transmembrane domain or a glycosylphosphatidylinositol anchor. Cubilin is composed of several structural elements: An amino-terminal stretch of 106 amino acids followed by eight EGF-like repeats and 27 CUB domains (first described in complement subcomponents C1r/ C1s, EGF-related sea urchin protein, and bone morphogenic protein-1), which most likely constitute the ligand-binding domains (7,11,12).…”

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confidence: 99%

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Contribution of Cubilin and Amnionless to Processing and Membrane Targeting of Cubilin–Amnionless Complex

Coudroy¹,

Gburek²,

Kozyraki³

et al. 2005

Journal of the American Society of Nephrology

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Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with cubilin. By expression in transfected polarized MDCK cells of AMN and several cubilin fragments, including a functional "mini" version of cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with cubilin and co-localized with cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the cubilin-AMN complex to the apical membrane: AMN interacts with the EGF domains of cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.

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“…Owing to the high affinity between the cubilin and the two proteins RAP and megalin (Birn et al 1997;Kristiansen et al 1999;Yammani et al 2001; Christensen and Birn 2002), we tested the implication of the LRPAP1 (RAP) or the LRP2 (megalin) in MGA1 in this latest family. Three microsatellite markers flanking the LRPAP1 gene and two markers flanking the LRP2 gene covering regions of 3 and 2 Mb, respectively, were investigated.…”

Section: Haplotype and Linkage Analysesmentioning

confidence: 99%

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

Bouchlaka

Maktouf

Mahjoub³

et al. 2007

J Hum Genet

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Megaloblastic anaemia 1 (MGA1) is a rare autosomal recessive condition characterized by selective intestinal vitamin B12 malabsorption and proteinuria. More than 200 MGA1 patients have been identified worldwide, but the disease is relatively prevalent in Finland, Norway and several Eastern Mediterranean regions. MGA1 is genetically heterogeneous and can be caused by mutations in either the cubilin (CUBN) or the amnionless (AMN) gene. In the present study we investigated the molecular defect underlying MGA1 in nine Tunisian patients belonging to six unrelated consanguineous families. Haplotype and linkage analyses, using microsatellite markers surrounding both CUBN and AMN genes, indicated that four out of the six families were likely to be linked to the CUBN gene. Patients from these families were screened for the Finnish, Mediterranean and Arabian mutations already published. None of the screened mutations could be detected in our population. One family showed a linkage to AMN gene. Direct screening of the AMN gene allowed the identification of the c.208-2A>G mutation, previously described in a Jewish Israeli patient of Tunisian origin and in Turkish patients. This suggests that the c.208-2A>G mutation may derive from a single Mediterranean founder ancestor. For the last family, haplotype analysis excluded both CUBN and AMN genes, suggesting the existence of a third locus that may cause MGA1.

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Identification and Characterization of Two Distinct Ligand Binding Regions of Cubilin

Cited by 43 publications

References 29 publications

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

Contribution of Cubilin and Amnionless to Processing and Membrane Targeting of Cubilin–Amnionless Complex

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

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