Molecular Dissection of the Intrinsic Factor-Vitamin B12 Receptor, Cubilin, Discloses Regions Important for Membrane Association and Ligand Binding

Abstract

Cubilin, the receptor for intrinsic factor-vitamin B 12 , is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B 12 -carrier complex, cubilin also binds receptor-associated protein. 1 depends on gastric intrinsic factor (IF), which undergoes a specific and strong complex formation with the vitamin in the intestinal lumen (1). The vitamin-carrier complex is subsequently internalized after being recognized by the high affinity membrane receptor, cubilin (2, 3). The binding of IF-B 12 is suggested to occur via the N-terminal region of IF (4) and only when B 12 is bound to the carrier protein (1-3, 5). The physiological importance of the specific B 12 carrier and its receptor is evident from the B 12 deficiency, characterized by the megaloblastic anemia and neurological symptoms related to defective functional expression of IF or cubilin (6 -11). In addition to binding IF-B 12 , cubilin also binds receptor-associated protein (RAP) (5), and growing evidence suggests that the receptor has other biological functions. This is in particular indicated by the fact that kidney and yolk sac epithelia exhibit a high cubilin expression (2, 5, 12), albeit IF is synthesized in the gastric epithelium and virtually only is present in the gastrointestinal lumen.The recent determination of the primary structure of the rat and human cubilin molecule (13, 14) revealed an ϳ3600-aa protein with a short N-terminal segment followed by 8 epidermal growth factor (EGF) repeats and a large cluster of 27 CUB 2 domains. No classical transmembrane segment has been predicted, and the overall structure is distinct from known endocytic receptors, including the RAP-binding members of the low density lipoprotein (LDL) receptor family (15). Two mutations, one causing a Pro/Leu substitution in CUB domain 8 and another suggested to activate an intronic cryptic splice site leading to truncation of the receptor in CUB domain 6, have recently been identified in the cubilin gene of patients with inherited B 12 malabsorption (11).To link the structural and functional features of cubilin, we established a comprehensive panel of transfected Chinese hamster ovary (CHO) cells expressing various segments of the receptor. Biochemical analyses of the cells and expression products now delineate the regions important for membrane association and binding of IF-B 12 and RAP. EXPERIMENTAL PROCEDURESLigands and Receptors-Porcine and human IF-B 12 were purified from gastric mucosa extract as described (16). Human RAP was produced as a recombinant protein in Escherichia coli. Rabbit cubilin was purified from renal cortex by IF-B 12 and RAP affinity chromatography, as described previously (5). Construction of Plasmids for Expression of Recombinant Rat CubilinFragments-Cubilin cDNA fragments extended with enzyme restriction sites were amplified by polymerase c...

Crystal structure of human intrinsic factor: Cobalamin complex at 2.6-Å resolution

“…CUB is a large multidomain protein (460 kDa), which contains 27 tandem CUB domains that harbor the IF-Cbl-binding site (34,35). It has been proposed that a site on IF for binding the CUB receptor might lie within the segment between residues 25 and 62 (29).…”

Identification and Characterization of Two Distinct Ligand Binding Regions of Cubilin

“…3). The N terminus of cubilin has also been demonstrated to form coiled-coil ␣-helixes (17) that are amphipathic in nature (14) and are thought to interact with the lipid bilayer. The amphipathic nature of the 113-residue N terminus is evident as it formed aggregates following its synthesis (Fig.…”

“…Moreover, when this region along with the EGF-like repeats was stably expressed in Chinese hamster ovary cells, it was retained within the cell unlike other fragments of cubilin, which were all secreted efficiently. However, the ligand binding ability of this fraction was not studied (14). Thus, based on the ability of this fragment to bind the ligands and its hydrophobic nature, the N-terminal region of cubilin may interact with the outer leaflet of the apical bilayer membrane.…”

“…Based on the recent elucidation of its sequence, IFCR is now renamed cubilin mainly because it consists of a contiguous stretch of 27 CUB domains that represent nearly 88% of its total mass of 460 kDa and because IF-Cbl binding has been shown to be localized to a region from CUB domain 5 to CUB domain 8 (14). A CUB domain is a 110 -115-amino acid module present in developmentally regulated proteins that is known to form a ␤-barrel.…”

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

“…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.…”