Contribution of Cubilin and Amnionless to Processing and Membrane Targeting of Cubilin–Amnionless Complex Gwe ´nae ¨lle Coudroy,* Jakub Gburek,* ‡ Renata Kozyraki,* Mette Madsen, § Germain Trugnan, † Søren K. Moestrup, § Pierre J. Verroust,* and Miche `le Maurice* *INSERM-U538, Saint-Antoine Medical Faculty, Paris, France; † Universite ´ Pierre et Marie Curie, Paris, France; ‡ Department of Pharmaceutical Biochemistry, University of Wroclaw, Wroclaw, Poland; and § Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark 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. J Am Soc Nephrol 16: 2330 –2337, 2005. doi: 10.1681/ASN.2004110925 C ubilin, which is also known as the intrinsic factor- cobalamin (IF-B12) receptor (1), is a multiligand recep- tor expressed at the apical plasma membrane and in endocytic compartments of several absorptive epithelia, espe- cially the kidney proximal tubule, the small intestine, and the visceral yolk sac, where it plays a scavenger receptor function, in concert with megalin (2–5). Mutations in the cubilin gene have been identified in a group of patients who have Imer- slund-Gra ¨sbeck’s syndrome (or megaloblastic anemia type 1) (6,7), a rare genetic disorder characterized by defective intesti- nal absorption of vitamin B 12 and proteinuria. The latter symp- tom is explained by the role of cubilin in the uptake of filtered plasma proteins including the vitamin D binding protein, albu- min, hemoglobin, transferrin, Ig light chains, Clara cell secre- tory protein, and apolipoprotein A1. Cubilin is a highly conserved membrane glycoprotein of 460 kD that has been characterized in the rat (8), human (9), and dog (10). The protein contains a cleavable signal sequence that allows the polypeptide chain to enter the endoplasmic reticu- lum (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 do- mains (7,11,12). Although cubilin is physiologically expressed at the apical pole of selected epithelial cells, there is at present no informa- tion on its trafficking in polarized cells. Furthermore, the mech- anism of membrane anchoring of cubilin is still unclear. Previ- ous studies based on the expression of structural elements of cubilin in CHO cells suggested that the amino-terminal domain is necessary for membrane anchoring of the receptor (11). This region contains a putative amphipathic helix with conserved clustered hydrophobic residues, but it has not been shown whether this region is embedded into the lipid bilayer of the plasma membrane. However, this hypothesis was recently called into question with the demonstration that cubilin puri- fies as a complex with amnionless (AMN) (13), a 45- to 50-kD transmembrane protein that is highly expressed in both small intestine and kidney and that is mutated in patients with de- fective absorption of vitamin B 12 but otherwise normal cubilin gene (14). In CHO cells, AMN was necessary and sufficient to allow a functional mini version of cubilin to traffic to the cell surface and internalize IF-B12 (13). That cubilin functions as a complex with AMN has important implications for cubilin trafficking and possibly membrane as- Received November 10, 2004. Accepted May 16, 2005. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Pierre J. Verroust, INSERM U538, Centre Hos- pitalier Universitaire Saint Antoine, 27 Rue Chaligny, 75012 Paris, France. Phone: 33-1-4001-1323; Fax: 33-1-4001-1390; E-mail: verroust@ccr.jussieu.fr Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1608-2330