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The Intrinsic Factor-Vitamin B12 Receptor and Target of Teratogenic Antibodies Is a Megalin-binding Peripheral Membrane Protein with Homology to Developmental Proteins*

Open AccessPublished:February 27, 1998DOI:https://doi.org/10.1074/jbc.273.9.5235
      The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.
      Intestinal uptake of vitamin B12(B12)
      The abbreviations used are: B12, vitamin B12; BN cell, yolk sac-derived mouse sarcoma virus-transformed Brown Norway rat epithelial cell; EGF, epidermal growth factor; IF, intrinsic factor; RAP, receptor-associated protein; RACE, rapid amplification of cDNA ends; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s).
      ,
      Vitamin B12 is cyanocobalamin. In the organism, cyanocobalamin is converted to the active forms of cobalamin, methyl- and 5′-deoxyadenosylcobalamin. The abbreviation B12 is employed to cover all forms of cobalamin that can be converted to the active form.
      is facilitated by a receptor recognizing B12 in complex with gastric intrinsic factor (IF) (for a review, see Ref.
      • Seetharam B.
      • Seetharam S.
      • Li N.
      • Ramanujam K.S.
      ). Failure of either IF production or receptor expression leads inevitably to a B12 deficiency state and disease (
      • Yang Y.M.
      • Duclos R.
      • Rosenberg A.J.
      • Catrou P.G.
      • Levine J.S.
      • Podell E.R.
      • Allen R.H.
      ,
      • Gueant J.L.
      • Saunier M.
      • Gastin I.
      • Safi A.
      • Lamireau T.
      • Duclos B.
      • Bigard M.A.
      • Gräsbeck R.
      ). Although the existence of a receptor for IF-B12 was recognized nearly 3 decades ago (
      • Rothenberg S.P.
      • Huhti A.L.
      ), its biochemical properties and structure have remained elusive, possibly because of difficulties in purifying it in high amounts from the terminal ileum (
      • Seetharam B.
      • Alpers D.H.
      • Allen R.H.
      ,
      • Cotter R.
      • Rothenberg S.P.
      ). The observation by Seetharamet al. (
      • Seetharam B.
      • Levine J.S.
      • Ramasamy M.
      • Alpers D.H.
      ) that the receptor was expressed in considerably greater amounts in the kidney and yolk sac facilitated the production of antibodies inhibiting IF-B12 binding (
      • Seetharam B.
      • Christensen E.I.
      • Moestrup S.K.
      • Hammond T.G.
      • Verroust P.J.
      ,
      • Birn H.
      • Verroust P.J.
      • Nexø E.
      • Hager H.
      • Jacobsen C.
      • Christensen E.I.
      • Moestrup S.K.
      ) and confirmed the receptor as a glycoprotein of >200 kDa. We have recently estimated a size of 460 kDa of the IF-B12 affinity-purified receptor (
      • Birn H.
      • Verroust P.J.
      • Nexø E.
      • Hager H.
      • Jacobsen C.
      • Christensen E.I.
      • Moestrup S.K.
      ).
      Since IF is only detected at very low levels in the circulation and in nongastrointestinal tissues (
      • Seetharam B.
      • Seetharam S.
      • Li N.
      • Ramanujam K.S.
      ), the high expression of the receptor in kidney and yolk sac may suggest that it has other functions/ligands. This is further indicated by several lines of evidence. First, patients with hereditary intestinal malabsorption of IF-B12, known as Imerslund-Gräsbeck syndrome (
      • Imerslund O.
      ,
      • Gräsbeck R.
      • Gordin R.
      • Kantero I.
      • Kuhlbäck B.
      ), have proteinuria, indicating that the receptor facilitating IF-B12 uptake in the intestine is also important for normal kidney function. The significance of this observation is reinforced by the presence of proteinuria in a family of dogs that synthesize a nonfunctional receptor (
      • Fyfe J.C.
      • Ramanujam K.S.
      • Ramaswamy K.
      • Patterson D.F.
      • Seetharam B.
      ). Second, we have recently shown that the receptor binds RAP to a site distinct from the binding site for IF-B12(
      • Birn H.
      • Verroust P.J.
      • Nexø E.
      • Hager H.
      • Jacobsen C.
      • Christensen E.I.
      • Moestrup S.K.
      ). RAP is a chaperone-like protein that protects multiple ligand binding sites of processed low density lipoprotein receptor family proteins, in particular the two giant receptors megalin and low density lipoprotein receptor-related protein (
      • Willnow T.E.
      • Rohlmann A.
      • Horton J.
      • Otani H.
      • Braun J.R.
      • Hammer R.E.
      • Herz J.
      ,
      • Bu G.
      • Geuze H.J.
      • Strous G.J.
      • Schwartz A.L.
      ). Finally, immunopathological studies indicate a key role of the IF-B12-binding protein in embryonic development. We have recently demonstrated that the target antigen (initially designated “gp280”) of teratogenic antibodies (
      • Brent R.L.
      • Averich E.
      • Drapiewski V.A.
      ) was associated with the endocytic pathway of yolk sac epithelial cells (
      • Sahali D.
      • Mulliez N.
      • Chatelet F.
      • Dupuis R.
      • Ronco P.
      • Verroust P.
      ,
      • Le Panse S.
      • Ayani E.
      • Nielsen S.
      • Ronco P.
      • Verroust P.
      • Christensen E.I.
      ) and identical to the IF-B12 receptor (
      • Seetharam B.
      • Christensen E.I.
      • Moestrup S.K.
      • Hammond T.G.
      • Verroust P.J.
      ).
      The present study provides novel molecular insight into the multifaceted IF-B12 receptor structure and membrane interactions. Determination of its primary structure revealed a modular 3603-amino acid sequence characterized by the presence of a hitherto unreported cluster of 27 CUB
      The name CUB, introduced by Bork and Beckmann (
      • Bork P.
      • Beckmann G.
      ), is an abbreviation of complement subcomponents C1r/C1s, Uegf, Bmp1.
      3The name CUB, introduced by Bork and Beckmann (
      • Bork P.
      • Beckmann G.
      ), is an abbreviation of complement subcomponents C1r/C1s, Uegf, Bmp1.
      domains, which constitute multiple potential sites for interaction with proteins, carbohydrates, and phospholipids. Furthermore, the receptor lacks a transmembrane domain and cytoplasmic tail, although it is internalized and recycles (
      • Birn H.
      • Verroust P.J.
      • Nexø E.
      • Hager H.
      • Jacobsen C.
      • Christensen E.I.
      • Moestrup S.K.
      ,
      • Le Panse S.
      • Ayani E.
      • Nielsen S.
      • Ronco P.
      • Verroust P.
      • Christensen E.I.
      ,
      • Hammond T.G.
      • Verroust P.J.
      ,
      • Le Panse S.
      • Galcéran M.
      • Pontillon F.
      • Lelongt B.
      • van de Putte M.
      • Ronco P.M.
      • Verroust P.J.
      ). Direct binding of ligand affinity-purified IF-B12 receptor to megalin and colocalization at the ultrastructural level of the two proteins indicate that the internalization of the receptor may, at least in part, occur by interaction with megalin. Due to the predominant content of CUB domains, the receptor is here designated cubilin.

      Note Added in Proof

      The crystal solution structure of the CUB domain of spermadhesion has now been published (Romero, A., Romao, M. J. Varela, P. F., Kolln, I., Dias, J. M., Carvalho, A. L., Sanz, L., Topfer-Petersen, E., and Calvete, J. J. (1997) Nat. Struct. Biol. 10,783–788.

      Acknowledgments

      Technical assistance by Merete Grøfte, Kirsten Lassen, Marthé Galcéran, and Hanne Sidelmann is gratefully acknowledged. We thank Christian Jacobsen for helping with the analysis of the BIAcore data; Ebba Nexø and Jørgen Gliemann for continuous support and helpful suggestions; and Pierre Fouret and D. Sahali for assistance in the initiation of the project.

      References

        • Seetharam B.
        • Seetharam S.
        • Li N.
        • Ramanujam K.S.
        Bhatt H.R. James V.H.T. Besser G.M. Bottazzo G.F. Keen H. Advances in Thomas Addisons's Diseases. II. 1994: 393-406 (Journal of Endocrinology Ltd., Almondsbury, Bristol, United Kingdom)
        • Yang Y.M.
        • Duclos R.
        • Rosenberg A.J.
        • Catrou P.G.
        • Levine J.S.
        • Podell E.R.
        • Allen R.H.
        J. Clin. Invest. 1985; 76: 2057-2065
        • Gueant J.L.
        • Saunier M.
        • Gastin I.
        • Safi A.
        • Lamireau T.
        • Duclos B.
        • Bigard M.A.
        • Gräsbeck R.
        Gastroenterology. 1995; 108: 1622-1628
        • Rothenberg S.P.
        • Huhti A.L.
        J. Clin. Invest. 1968; 47: 913-923
        • Seetharam B.
        • Alpers D.H.
        • Allen R.H.
        J. Biol. Chem. 1981; 256: 3785-3790
        • Cotter R.
        • Rothenberg S.P.
        Br. J. Haematol. 1976; 34: 477-487
        • Seetharam B.
        • Levine J.S.
        • Ramasamy M.
        • Alpers D.H.
        J. Biol. Chem. 1988; 263: 4443-4449
        • Seetharam B.
        • Christensen E.I.
        • Moestrup S.K.
        • Hammond T.G.
        • Verroust P.J.
        J. Clin. Invest. 1997; 99: 2317-2322
        • Birn H.
        • Verroust P.J.
        • Nexø E.
        • Hager H.
        • Jacobsen C.
        • Christensen E.I.
        • Moestrup S.K.
        J. Biol. Chem. 1997; 272: 26497-26504
        • Imerslund O.
        Acta Paediatr. Scand. 1960; 49: 1-115
        • Gräsbeck R.
        • Gordin R.
        • Kantero I.
        • Kuhlbäck B.
        Acta Med. Scan. 1960; 167: 289-296
        • Fyfe J.C.
        • Ramanujam K.S.
        • Ramaswamy K.
        • Patterson D.F.
        • Seetharam B.
        J. Biol. Chem. 1991; 266: 4489-4494
        • Willnow T.E.
        • Rohlmann A.
        • Horton J.
        • Otani H.
        • Braun J.R.
        • Hammer R.E.
        • Herz J.
        EMBO J. 1996; 15: 2632-2639
        • Bu G.
        • Geuze H.J.
        • Strous G.J.
        • Schwartz A.L.
        EMBO J. 1995; 14: 2269-2280
        • Brent R.L.
        • Averich E.
        • Drapiewski V.A.
        Proc. Soc. Exp. Biol. Med. 1961; 106: 523-526
        • Sahali D.
        • Mulliez N.
        • Chatelet F.
        • Dupuis R.
        • Ronco P.
        • Verroust P.
        J. Exp. Med. 1988; 167: 213-218
        • Le Panse S.
        • Ayani E.
        • Nielsen S.
        • Ronco P.
        • Verroust P.
        • Christensen E.I.
        Eur. J. Cell Biol. 1997; 72: 257-267
        • Hammond T.G.
        • Verroust P.J.
        Am. J. Physiol. 1994; 266: F554-F562
        • Le Panse S.
        • Galcéran M.
        • Pontillon F.
        • Lelongt B.
        • van de Putte M.
        • Ronco P.M.
        • Verroust P.J.
        Eur. J. Cell Biol. 1995; 67: 120-129
        • Moestrup S.K.
        • Nielsen S.
        • Andreasen P.
        • Jorgensen K.E.
        • Nykjaer A.
        • Røigaard H.
        • Gliemann J.
        • Christensen E.I.
        J. Biol. Chem. 1993; 268: 16564-16570
        • Sahali D.
        • Mulliez N.
        • Chatelet F.
        • Laurent-Winter C.
        • Citadelle D.
        • Sabourin J.C.
        • Roux C.
        • Ronco P.
        • Verroust P.
        Am. J. Pathol. 1993; 142: 1654-1667
        • Nexø E.
        • Olesen H.
        • Bucher D.
        • Thomsen J.
        Biochim. Biophys. Acta. 1977; 494: 395-402
        • Jensen P.H.
        • Gliemann J.
        • Ørntoft T.
        FEBS Lett. 1992; 29: 129-132
        • Christensen E.I.
        • Nielsen S.
        • Moestrup S.K.
        • Borre C.
        • Maunsbach A.B.
        • de Heer E.
        • Hammond T.G.
        • Verroust P.
        Eur. J. Cell Biol. 1995; 66: 349-364
        • Moestrup S.K.
        • Birn H.
        • Fischer P.B.
        • Petersen C.M.
        • Verroust P.
        • Sim R.B.
        • Christensen E.I.
        • Nexø E.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8612-8617
        • Selander-Sunnerhagen M.
        • Ullner M.
        • Persson E.
        • Teleman O.
        • Stenflo J.
        • Drakenberg T.
        J. Biol. Chem. 1992; 267: 19642-19649
        • Bork P.
        • Beckmann G.
        J. Mol. Biol. 1993; 231: 539-545
        • Levy D.
        Essays Biochem. 1996; 31: 49-59
        • Saito A.
        • Pietromonaco S.
        • Loo A.K.
        • Farquhar M.G.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9725-9729
        • Calvete J.J.
        • Carrera E.
        • Sanz L.
        • Topfer-Petersen E.
        Biol. Chem. 1996; 377: 521-527
        • Dostalova Z.
        • Calvete J.J.
        • Topfer-Petersen E.
        Biol. Chem. Hoppe-Seyler. 1995; 376: 237-242
        • Petersen C.M.
        • Nielsen M.S.
        • Nykjaer A.
        • Jacobsen L.
        • Tommerup N.
        • Rasmussen H.H.
        • Røigaard H.
        • Gliemann J.
        • Madsen P.
        • Moestrup S.K.
        J. Biol. Chem. 1997; 272: 3599-3605
        • Menendez M.
        • Gasset M.
        • Laynez J.
        • Lopez-Zumel C.
        • Usobiaga P.
        • Topfer-Petersen E.
        • Calvete J.J.
        Eur. J. Biochem. 1995; 234: 887-896
        • Romero A.
        • Varela P.F.
        • Sanz L.
        • Topfer-Petersen E.
        • Calvete J.J.
        FEBS Lett. 1996; 382: 15-17
        • Dias J.M.
        • Carvalho A.L.
        • Kolln I.
        • Calvete J.J.
        • Topfer-Petersen E.
        • Varela P.F.
        • Romero A.
        • Urbanke C.
        • Romao M.J.
        Protein Sci. 1997; 6: 725-727
        • Thiel S.
        • Vorup-Jensen T.
        • Stover C.M.
        • Schwaeble W.
        • Laursen S.B.
        • Poulsen K.
        • Willis A.C.
        • Eggleton P.
        • Hansen S.
        • Holmskov U.
        • Reid K.B.
        • Jensenius J.C.
        Nature. 1997; 386: 506-510
        • Busby T.F.
        • Ingham K.C.
        Biochemistry. 1990; 29: 4613-4618
        • Calvete J.J.
        • Raida M.
        • Gentzel M.
        • Urbanke C.
        • Sanz L.
        • Topfer-Petersen E.
        FEBS Lett. 1997; 407: 201-206
        • Davis G.G.
        New Biol. 1990; 2: 410-419
        • Rao Z.
        • Handford P.
        • Mayhew M.
        • Knott V.
        • Brownlee G.G.
        • Stuart D.
        Cell. 1995; 82: 131-141
        • Leung C.C.
        J. Exp. Med. 1982; 156: 372-384
        • Nykjaer A.
        • Conese M.
        • Christensen E.I.
        • Olson D.
        • Cremona O.
        • Gliemann J.
        • Blasi F.
        EMBO J. 1997; 16: 2610-2620
        • Herz J.
        • Clouthier D.E.
        • Hammer R.E.
        Cell. 1992; 71: 411-421
        • Dan N.
        • Cutler D.F.
        J. Biol. Chem. 1994; 269: 18849-18855
        • Iadonato S.P.
        • Bu G.
        • Maksymovitch E.A.
        • Schwartz A.L.
        Biochem. J. 1993; 296: 867-875
        • Beckman D.A.
        • Lloyd J.B.
        • Brent R.L.
        Int. J. Dev. Biol. 1997; 41: 315-318
        • Lloyd J.B.
        Teratology. 1990; 41: 383-393
        • Le Panse S.
        • Ayani E.
        • Mulliez N.
        • Chatelet F.
        • Cywiner-Golenzer C.
        • Galcéran M.
        • Citadelle D.
        • Roux C.
        • Ronco P.
        • Verroust P.
        Am. J. Pathol. 1994; 145: 1526-1536
        • Llirbat B.
        • Wolf C.
        • Chevy F.
        • Citadelle D.
        • Bereziat
        • Roux C.
        J. Lipid Res. 1997; 38: 22-34
        • Porter J.A.
        • Young K.E.
        • Beachy P.A.
        Science. 1996; 274: 255-259
        • Willnow T.E.
        • Hilpert J.
        • Armstrong S.A.
        • Rohlmann A.
        • Hammer R.E.
        • Burns D.K.
        • Herz J.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8460-8464
        • Herz J.
        • Willnow T.E.
        • Farese R.V.
        Nat. Genet. 1997; 15: 122-123