Identification and Characterization of Two Distinct Ligand Binding Regions of Cubilin
2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês
10.1074/jbc.m106419200
ISSN1083-351X
AutoresRaghunatha R. Yammani, Shakuntla Seetharam, Bellur Seetharam,
Tópico(s)Cellular transport and secretion
ResumoUsing polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6–8 bound both intrinsic factor-cobalamin (vitamin B12; Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110–115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca2+ dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60–65%. However, a combination of CUB domains 7 and 8 or 6–8 was needed to demonstrate significant Ca2+ dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6–8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds. Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6–8 bound both intrinsic factor-cobalamin (vitamin B12; Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110–115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca2+ dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60–65%. However, a combination of CUB domains 7 and 8 or 6–8 was needed to demonstrate significant Ca2+ dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6–8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds. cobalamin (vitamin B12) epidermal growth factor intrinsic factor intrinsic factor-cobalamin receptor polymerase chain reaction base pairs polyacrylamide gel electrophoresis The gastrointestinal uptake of cobalamin (vitamin B12; Cbl)1 occurs bound to gastric intrinsic factor (IF) by receptor-mediated endocytosis via a cell surface receptor, intrinsic factor-cobalamin receptor (IFCR) (1Seetharam B. Annu. Rev. Nutr. 1999; 19: 173-195Crossref PubMed Scopus (69) Google Scholar). IFCR isolated from canine ileal mucosa was estimated to have a molecular mass around 220 kDa and bound IF-Cbl with high affinity (2Seetharam B. Alpers D.H. Allen R.H. J. Biol. Chem. 1981; 256: 3785-3790Abstract Full Text PDF PubMed Google Scholar), and further proteolysis of this receptor revealed that IF-Cbl binding occurred with a number of receptor fragments of molecular masses as low as 80 kDa (3Seetharam B. Bagur S.S. Alpers D.H. J. Biol. Chem. 1982; 257: 183-189Abstract Full Text PDF PubMed Google Scholar). In addition to the intestinal ileal mucosa, very high levels of IFCR were also detected in mammalian kidney (4Seetharam B. Levine J.S. Ramasamy M. Alpers D.H. J. Biol. Chem. 1988; 263: 4443-4449Abstract Full Text PDF PubMed Google Scholar) and in rat yolk sac (5Ramanujam K.S. Seetharam S. Seetharam B. Biochim. Biophys. Acta. 1993; 1146: 243-246Crossref PubMed Scopus (19) Google Scholar). In contrast to canine ileal mucosal IFCR, the purified rat renal IFCR demonstrated a molecular mass of 457 kDa based on its amino acid and amino sugar content. It bound 2 mol of IF-Cbl (4Seetharam B. Levine J.S. Ramasamy M. Alpers D.H. J. Biol. Chem. 1988; 263: 4443-4449Abstract Full Text PDF PubMed Google Scholar) and like the yolk sac IFCR (5Ramanujam K.S. Seetharam S. Seetharam B. Biochim. Biophys. Acta. 1993; 1146: 243-246Crossref PubMed Scopus (19) Google Scholar) was developmentally regulated (6Seetharam S. Ramanujam K.S. Seetharam B. J. Biol. Chem. 1992; 267: 7421-7427Abstract Full Text PDF PubMed Google Scholar). Although the physiological significance for the high levels of IFCR expression in the kidney was not known for a number of years, using proximal tubular polarized epithelial opossum kidney cells it was demonstrated that the apically expressed IFCR was able to internalize IF-Cbl and mediate Cbl transport from the apical to basolateral medium (7Ramanujam K.S. Seetharam S. Dahms N. Seetharam B. J. Biol. Chem. 1991; 266: 13135-13140Abstract Full Text PDF PubMed Google Scholar). A later study using a canine model with selective inherited intestinal Cbl malabsorption syndrome (8Fyfe J.C. Ramanujam K.S. Ramaswamy K. Patterson D.F. Seetharam B. J. Biol. Chem. 1991; 266: 4489-4494Abstract Full Text PDF PubMed Google Scholar) demonstrated that in these animals the apical brush border membrane levels of IFCR in both ileal mucosa and kidney were depleted suggesting that the IFCR expressed in both tissues was a product of the same gene. Although it was thought for the last decade that the renal IFCR may have some other function, its structure was only recently delineated (9Moestrup S.K. Kozyraki R. Kristiansen M. Kaysen J.H. Rasmussen H.H. Brault D. Pontillon F. Goda F.O. Christensen E.I. Hammond T.G. Verroust P.J. J. Biol. Chem. 1998; 273: 5235-5242Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and a number of subsequent studies have shown that the renal IFCR is indeed a multifunctional receptor that is able to bind a variety of protein ligands with differing affinities. These include IF-Cbl (1–2 nm) (4Seetharam B. Levine J.S. Ramasamy M. Alpers D.H. J. Biol. Chem. 1988; 263: 4443-4449Abstract Full Text PDF PubMed Google Scholar), albumin (0.63 μm) (10Birn H. Fyfe J.C. Jacobsen C. Mounier F. Verroust P.J. Osskov H. Willnow T.E. Moestrup S.K. Christensen E.I. J. Clin. Invest. 2000; 105: 1353-1361Crossref PubMed Scopus (262) Google Scholar), high density lipoprotein (0.1 and 56 nm) (11Kozyraki R. Fyfe J. Kristiansen M. Gerdes C. Jacobsen C. Cui S. Christensen E.I. Aminoff M. de la Chapelle A. Krahe R. Verroust P.J. Moestrup S.K. Nat. Med. 1999; 5: 656-661Crossref PubMed Scopus (226) Google Scholar), and κ and γ light chains (160 and 12 mm) (12Batuman V. Driesbach A.W. Cyran J. Am. J. Physiol. 1990; 258: F1259-F1265PubMed Google Scholar, 13Batuman V. Verroust P.J. Navar G.L. Kaysen J.H. Goda F.O. Campbell W.C. Simon E. Pontillon F. Lyles M. Bruno J. Hammond T.G. Am. J. Physiol. 1998; 275: F246-F254PubMed Google Scholar). The presence of low and high affinity binding sites for high density lipoprotein and light chain suggested that there are at least two binding sites for these ligands. 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 (14Kristiansen M. Kozyraki R. Jacobsen C. Nexo E. Verroust P.J. Moestrup S.K. J. Biol. Chem. 1999; 274: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A CUB domain is a 110–115-amino acid module present in developmentally regulated proteins that is known to form a β-barrel. The acronym CUB is derived from proteinscomplement Clr/Cls, Uegf, and bone morphogenic protein-1 that have these domains. Upstream of these CUB domains, the rest of the cubilin molecule contains a 113-residue N-terminal region followed by eight EGF-like repeats. Consistent with these structural observations, the new name of cubilin will be used instead of IFCR throughout the rest of the manuscript. Despite these studies many aspects of the structure-function relationship of cubilin are poorly understood, particularly its ability to function as a multifunctional receptor, and the current studies were directed in addressing some of these issues. The results of the current studies show that cubilin contains two distinct ligand binding regions, one the 113-residue N terminus and the other CUB domains 7 and 8 that bind both IF-Cbl and albumin. Binding of both these ligands to each of these regions can be distinguished and is regulated by the formation of local disulfide bond(s) that form spontaneously in the absence of molecular chaperones and exogenously added oxidized glutathione. Sepharose and rat serum albumin were purchased from Sigma-Aldrich. pSec Tag B vector was from Invitrogen (Carlsbad, CA), and canine pancreatic microsomes and the TNT quick coupled transcription/translation system were from Promega (Madison, WI). Fluoro-HanceTM used for autoradiography was obtained from Research Products International Corp. (Mount Prospect, IL). Rat gastric intrinsic factor used in the current studies was purified from the rat stomach as described previously (15Seetharam B. Bakke J.E. Alpers D.H. Biochem. Biophys. Res. Commun. 1983; 115: 238-244Crossref PubMed Scopus (31) Google Scholar). Antiserum to purified rat renal cubilin raised in rabbits was prepared as described previously (4Seetharam B. Levine J.S. Ramasamy M. Alpers D.H. J. Biol. Chem. 1988; 263: 4443-4449Abstract Full Text PDF PubMed Google Scholar). Polyclonal antiserum to human EGF raised in rabbits was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Carrier-free Na125I for iodination of rat serum albumin and35S-Express protein labeling mix from PerkinElmer Life Sciences. IODO-GEN was from Pierce. Various cubilin fragments were amplified by polymerase chain reaction (PCR). The amplified products were subcloned into the expression vector pSec Tag B. The following 13 constructs were subcloned and expressed: N terminus (113 residues) (bp 3–14, bp 369–340), N-EGF (bp 3–14, bp 1404–1420), N-EGF + CUB 1–2 (bp 3–14, bp 2104–1221), CUB 1–4 (bp 4120–4136, bp 2777–2793), CUB 9–12 (bp 4171–4188, bp 5530–5556) CUB 12–17 (bp 5212–5229, bp 7338–7353), CUB 18–27 (bp 7354–7369, bp 10852–10869), CUB 5 (bp 2794–2808, bp 3124–3141), CUB 6 (bp 3142–3156, bp 3476–3492), CUB 7 (bp 3493–3507, bp 3816–3831), CUB 8 (bp 3832–3846, bp 4153–4170), CUB 7–8 (bp 3453–3507, bp 4153–4170), and CUB 6–8 (bp 3142–3156, bp 4153–4170). The sequence of each PCR product obtained was confirmed to represent the correct amplified sequence prior to its use. The plasmids were transcribed and translated in vitro by TNT quick coupled transcription and translation system from Promega. The 35S-translated products were then used for further characterization. The constructs were transcribed and translated by the TNT quick coupled system as described by the manufacturer. In some experiments canine pancreatic microsomal membranes (1 μl) were added to study the cotranslational processing and post-translational modification of 35S-labeled rat cubilin fragments. To confirm the ability of the added pancreatic microsomes to carry out core N-glycosylation, mRNA encoding α-factor and, for processing, mRNA encoding pre-β-lactamase provided by the manufacturer were used as controls. Rat serum albumin was coupled to CNBr-activated Sepharose, and rat gastric IF was coupled to Cbl-Sepharose. One milliliter of a 1:1 suspension in 10 mm Tris-HCl buffer, pH 7.4, of the Sepharose-linked ligands (albumin or IF-Cbl) was capable of binding to at least 500–700 ng of purified renal cubilin. The 35S-translated cubilin fragments (50,000–75,000 dpm) were incubated with 500 μl of a 1:1 suspension of Sepharose beads in 10 mm Tris-HCl, pH 7.4, containing 10 mmCaCl2 or 10 mm EDTA. In some experiments, the labeled cubilin fragments were preincubated with 2–5 μl of polyclonal antiserum to either cubilin or EGF. For experiments shown in Figs. 7 and 8 the addition of 5 μl of the polyclonal antiserum to cubilin and EGF resulted in maximum inhibition of ligand binding, and the addition of a higher amount (10–25 μl) of the antisera did not inhibit ligand binding further. Using similar amounts (50,000 dpm) of protein synthesized from constructs CUB 7, CUB 8, or CUB 6–8, 5 μl of antiserum was sufficient to precipitate >90% of all three labeled proteins. Competition of ligand binding was carried out similarly except that the labeled cubilin fragments were preincubated for 60 min at 22 °C with either rat IF-Cbl (50 ng) or rat serum albumin (100 ng) prior to their binding to Sepharose linked to albumin or rat IF-Cbl, respectively. Following binding for 60 min, the beads were exhaustively washed with 10 mm Tris-HCl, pH 7.4, containing 140 mm NaCl (15–20 ml), and radioactivity bound was then released by boiling the beads with SDS sample buffer and subjected to nonreducing SDS-PAGE.Figure 8SDS-PAGE of EGF antiserum-treated, affinity-purified N terminus cubilin fraction. Prior to purification, the translated 35S-labeled 113-residue N terminus + eight EGF-like repeats was treated with 5 μl of EGF antiserum for 60 min at 22 °C and then subjected to ligand affinity chromatography as indicated. The bound radioactivity was eluted and subjected to SDS-PAGE, and the bands were visualized by fluorography. The data shown is a typical representation of three separate SDS-PAGE experiments from two separate competition experiments.View Large Image Figure ViewerDownload (PPT) 35S-Labeled translation products or the ligand affinity-purified cubilin fragments were subjected to nonreducing SDS-PAGE (12%). Gels were fixed and then treated with Fluoro-HanceTM for about 30 min (as described by the manufacturer), and the bands were visualized by fluorography. In some experiments, the labeled cubilin fragments were first reduced with 2-mercaptoethanol (2%) and then alkylated withN-ethylmaleimide (1 mm) prior to SDS-PAGE. The bands were quantified by the AMBIS radioimaging system. SDS-PAGE data shown in Figs. Figure 2, Figure 3, Figure 4 and Figs. 11 and 12 are typical representations of at least three separate translation and ligand binding experiments and reductive alkylation experiments, respectively.Figure 3SDS-PAGE analysis of 35S-labeled N-terminal cubilin fragments. In vitro transcribed and translated cubilin fragments, 113-residue N terminus + eight EGF-like + domains CUB 1 and 2 (lane 1) or the 113-residue N terminus alone (lane 2) (A) or the translated products bound to and eluted from Sepharose-IF-Cbl (lanes 1 and2) (B) or Sepharose-rat serum albumin columns (lanes 1 and 2) (C) were subjected to nonreducing SDS-PAGE, and the bands were visualized by fluorography. The position of the molecular weight markers (lane 0) (A) are shown.View Large Image Figure ViewerDownload (PPT)Figure 4SDS-PAGE analysis of 35S-labeled CUB domain (5Ramanujam K.S. Seetharam S. Seetharam B. Biochim. Biophys. Acta. 1993; 1146: 243-246Crossref PubMed Scopus (19) Google Scholar, 6Seetharam S. Ramanujam K.S. Seetharam B. J. Biol. Chem. 1992; 267: 7421-7427Abstract Full Text PDF PubMed Google Scholar, 7Ramanujam K.S. Seetharam S. Dahms N. Seetharam B. J. Biol. Chem. 1991; 266: 13135-13140Abstract Full Text PDF PubMed Google Scholar, 8Fyfe J.C. Ramanujam K.S. Ramaswamy K. Patterson D.F. Seetharam B. J. Biol. Chem. 1991; 266: 4489-4494Abstract Full Text PDF PubMed Google Scholar) fragments. The indicated CUB domains were translated (A), or the same fractions purified from Sepharose-IF-Cbl (B) or Sepharose-rat serum albumin columns (C) were subjected to nonreducing SDS-PAGE, and the bands were visualized by fluorography. The position of molecular weight markers (lane 0) and products of endogenous translation (lane 1) are shown.View Large Image Figure ViewerDownload (PPT)Figure 11SDS-PAGE of 35S-labeled 113-residue N terminus and the CUB 6–8 fractions following reductive alkylation. The nonreduced (lanes 1) or reduced and alkylated (lanes 2) translated cubilin fragments or the translated cubilin fragments purified from either Sepharose-IF-Cbl, nonreduced (lanes 3) or reduced (lanes 4), or Sepharose-rat albumin, nonreduced (lanes 5) or reduced (lanes 6), were subjected to SDS-PAGE. The bands were visualized by fluorography.View Large Image Figure ViewerDownload (PPT)Figure 12Reductive alkylation of CUB domains 5, 6, 7, and 8. The indicated translated 35S-labeled CUB domains were reduced, alkylated, and subjected to SDS-PAGE. Lane 1, unreduced; lane 2, reduced. Details are provided under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT) Rat renal apical membranes were isolated by the Ca2+ aggregation method as described previously (4Seetharam B. Levine J.S. Ramasamy M. Alpers D.H. J. Biol. Chem. 1988; 263: 4443-4449Abstract Full Text PDF PubMed Google Scholar). The Ca2+-dependent binding of ligand, IF-[57Co]Cbl (100–2500 pg), or 125I-rat serum albumin (10–200 ng) (specific activity, 200,000 dpm/μg of albumin) was determined using the rat renal membranes (25–50 μg of protein). The total ligand bound in the presence of 10 mmTris-HCl buffer, pH 7.4, containing 10 mm CaCl2was subtracted from that bound in the presence of the same buffer but containing 10 mm EDTA to obtain Ca2+-specific ligand binding. Sequence alignment of the N-terminal region and CUB domains 7 and 8 were performed by Jellyfish version 1.4 using matrix-Gonnet. The various regions of cubilin that were amplified by reverse transcriptase PCR are shown in Fig. 1. Initially all cubilin fragments were in vitro transcribed and translated to test for their ability to synthesize a functional protein. Earlier studies (14Kristiansen M. Kozyraki R. Jacobsen C. Nexo E. Verroust P.J. Moestrup S.K. J. Biol. Chem. 1999; 274: 20540-20544Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) had revealed that when the media collected from stably transfected cells were tested for IF-Cbl binding, only the media from cells transfected with CUB domains 5–8 bound IF-Cbl, and none of the other cubilin fragments demonstrated IF-Cbl binding activity. In our studies (Fig. 2), four cubilin fragments encompassing CUB domains 1–4, 9–12, 12–17, and 18–27 were efficiently translated in vitro (lanes 1) and produced major proteins of molecular masses 45, 47, 70, and 100 kDa, respectively, consistent with the number of CUB domains present in each fragment. Each CUB domain consists of 110–115 residues with an average polypeptide mass of around 110 kDa. The [35S]methionine-labeled proteins synthesized by these cubilin fragments failed to bind either IF-Cbl (lanes 2) or albumin (lanes 3). In contrast, the ∼71-kDa cubilin fragment synthesized (Fig. 3 A,lane 1) from the N-terminal fraction, which contained the 113-residue N-terminal region along with the eight EGF-like repeats and CUB 1-2 demonstrated Ca2+-dependent binding of both IF-Cbl (Fig. 3 B, lane 1) and albumin (Fig.3 C, lane 1). Within this region, the 113-residue N-terminal region, which synthesized a protein of ∼18 kDa (Fig. 3 A, lane 2) was by itself enough and sufficient to bind both IF-Cbl and albumin (Fig. 3, B andC, lanes 2). The cell-free translation of the 113-residue N terminus resulted in the synthesis of a predominant 18-kDa band, but there were three other bands of higher molecular mass (36, 39, and 44 kDa). These higher molecular mass forms may represent the aggregated forms of the 18-kDa form, and the size difference between them could be due to different amounts of Triton X-100 bound to them. This conclusion is based on the observation that all three forms bound both ligands. Prior incubation of the translated product with Sepharose alone did not eliminate these bands from the fraction that was eluted from ligand affinity matrix (data not shown). The DNA fragment encoding the eight EGF-like repeat region synthesized a protein of 45 kDa but did not bind either of the two ligands (data not shown). Since our earlier data (Fig. 2) had demonstrated that no ligand binding activity occurred with cubilin fragments synthesized downstream of CUB domain 9 and CUB domains 1–4, attention was focused to test ligand binding with cubilin fragments synthesized by CUB domains 5–8. While all of the individual CUB domains between 5 and 8 were able to synthesize protein products of molecular masses between 17 and 20 kDa (Fig. 4 A), only the proteins synthesized with CUB 7 and CUB 8 were able to bind both IF-Cbl (Fig.4 B) and albumin (Fig. 4 C). In addition, protein products of molecular mass 28 or 37 kDa synthesized from CUB 7-8 or 6–8, respectively, also bound both the ligands (Fig. 4, Band C). Lack of ligand binding (Fig. 4, B andC) observed by protein synthesized by CUB 5 or CUB 6 was not due to low levels of protein expressed by these constructs since the amount of 35S radioactivity used for affinity ligand binding was 3–4 times higher than that used for SDS-PAGE (Fig.4 A) analysis of the translated products. The two ligand binding regions of cubilin, the 113-residue N terminus, which included the eight EGF-like repeats along with CUB domains 1 and 2, and CUB domains 6–8 region contained three and five potentialN-glycosylation sites, respectively. Thus, we wanted to test whether these sites are utilized for N-glycosylation and if so, whether glycosylation at these site(s) affects ligand binding. Thus, these constructs were translated in vitro in the presence and absence of canine pancreatic microsomes. SDS-PAGE analysis (Fig. 5) failed to detect any shift in the electrophoretic mobility (Fig. 5 A) with either region 1 representing the N terminus + eight EGF-like repeats + CUB domains 1 and 2 (Fig. 5 A, compare mobility in lanes 3 and4) or region 2 containing CUB domains 6–8 (Fig.5 A, compare mobility in lanes 1 and2). Moreover, both regions of cubilin bound the ligands whether they were synthesized in the absence (Fig. 5, B andC, lanes 1) or the presence (Fig. 5, Band C, lanes 2) of pancreatic microsomes. Taken together, these observations suggested that under the experimental conditions used in the present studies these sites are notN-glycosylated and that the ligand binding is not affected when cell-free translation was carried out with the cotranslational addition of canine pancreatic microsomes. However, a positive control incubation to test the activity of the pancreatic microsomes indicated that under similar experimental conditions, Saccharomyces cerevisiae α-factor was processed with coreN-glycosylation (Fig. 5 A, compare mobilities oflanes 5 and 6) and that Escherichia coli β-lactamase is processed with cleavage of its signal peptide (Fig. 5 A, lanes 7 and8). These observations indicated that the lack of coreN-glycosylation of the cubilin fragments was not due to the use of an inactive sample of canine pancreatic microsomes. The binding of IF-Cbl to its receptor is Ca2+-dependent, but it is not known whether the dependence on Ca2+ for ligand binding is a property of the full-length receptor or can be demonstrated with functionally active regions of cubilin. To address this issue, the binding of 35S-labeled cubilin fragments were tested for Ca2+ dependence of binding to both IF-Cbl and albumin (Fig. 6). The binding of IF-Cbl and albumin to native renal brush border membrane was inhibited by EDTA by 97 and 50%, respectively. EDTA-inhibitable binding of IF-Cbl and albumin to region 1 encompassing only the 113-residue N-terminal region of cubilin was 64 and 70%, respectively. Interestingly, the inclusion of eight EGF-like repeats with the 113-residue N terminus resulted in the abolishment of Ca2+dependence for albumin binding, but the binding of IF-Cbl was 50% Ca2+-dependent. In contrast, both CUB domains 7 and 8 bound IF-Cbl and albumin but demonstrated only 5–8% Ca2+ dependence for the binding of both ligands. Although the total ligand bound (Fig. 6, dotted line) did not significantly change, CUB 7-8 or CUB 6–8 demonstrated an increased Ca2+ dependence for both ligands. Ca2+-dependent binding of both IF-Cbl and albumin to CUB 7-8 was about 50%. While inclusion of CUB 6 further increased the Ca2+ dependence of IF-Cbl binding by an additional 40–45% to almost 95%, it had no additional effect on Ca2+ dependence of albumin binding by CUB 7-8. Polyclonal antiserum to rat renal cubilin was able to inhibit by 58–64% (Fig. 7) IF-Cbl and albumin binding to the 113-residue N terminus. However, it inhibited by 10, 25, and 60% the binding of IF-Cbl to CUB 7, CUB 8, and CUB 6–8, respectively. On the other hand, the binding of albumin to CUB 7 and CUB 8 was inhibited between 55 and 60% and reached 95% for CUB 6–8 (Fig. 7). Studies with antiserum to EGF (Fig.8), which is relevant to only region 1, revealed inhibition of albumin binding by nearly 90%, while it had very little or no effect on IF-Cbl binding to this region. To examine whether the two protein ligands compete for binding, binding of one ligand was carried out after a preincubation of the35S-labeled cubilin fragments with the other ligand. Preincubation with excess albumin did not inhibit the binding of IF-Cbl by either the 113-residue or the CUB 6–8 fragment (Fig.9). On the other hand, when similar experiments were carried out with preincubation in the presence of IF-Cbl, the binding of albumin by both regions of cubilin was inhibited by >90–95%. Sequence analysis of the 113-residue N-terminal ligand binding region of cubilin revealed two cysteine residues (Fig.10) suggesting the potential formation of only one disulfide bond in this region. On the other hand, sequence alignment of the individual CUB domains 7 and 8 revealed (Fig. 10) the presence of four cysteine residues in each one of these CUB domains present at identical positions. To test whether local disulfide bonds were formed following translation of the two functional regions of cubilin and to further examine whether the disulfide bonding is required for ligand binding, the labeled translated fragments were subjected to reductive alkylation and ligand binding. SDS-PAGE analysis (Fig. 11) revealed that following reductive alkylation, the electrophoretic mobility of the translated 113-residue N-terminal region decreased (lane 2) relative to the unreduced sample (lane 1). In addition, while the nonreduced N-terminal protein bound both IF-Cbl (lane 3) and albumin (lane 5), reduction of the only disulfide bond formed between Cys-63 and Cys-117 destroyed binding of both ligands (lanes 4 and6). Formation of a disulfide bond(s) as revealed by decreased electrophoretic mobility was also evident with the cubilin fragment encoded by CUB 6–8 (lanes 1 and 2). Reductive alkylation inactivated binding of both IF-Cbl (lane 4) and albumin (lane 6) relative to the binding obtained with the nonreduced forms (lanes 3 and5). Reductive alkylation of these two cubilin regions synthesized in the presence or absence of canine pancreatic microsomes also revealed an identical shift in the mobility of the reduced forms of cubilin fragments (data not shown). To further confirm whether the loss of ligand binding due to disruption of disulfide bonding in region 2 (CUB 6–8) is due to loss of intra- or inter-CUB domain disulfide bonds, reductive alkylation of individual CUB domains was carried out. SDS-PAGE analysis of the labeled proteins revealed (Fig. 12) that CUB domains 7 and 8, which bind the ligand, demonstrated disulfide bonding, and upon reductive alkylation, ligand binding to both these CUB domains was completely inhibited (data not shown). However, CUB domains 5 and 6, which do not bind the ligand (Fig. 4), revealed the presence of disulfide bonding in CUB 5 but not CUB 6. These observations indicated that intramolecular disulfide bonds within CUB 7 or CUB 8 are important for ligand binding. Cubilin is a 460-kDa multidomain, multifunctional endocytic receptor expressed in the apical membranes of tissue epithelial cells and functions synergistically (16Christensen E.I. Birn H. Am. J. Physiol. 2001; 280: F562-F573Crossref PubMed Google Scholar) with megalin, another endocytic receptor of molecular mass 660 kDa. For an endocytic receptor, cubilin is unique in that it has no discernable transmembrane domain (9Moestrup S.K. Kozyraki R. Kristiansen M. Kaysen J.H. Rasmussen H.H. Brault D. Pontillon F. Goda F.O. Christensen E.I. Hammond T.G. Verroust P.J. J. Biol. Chem. 1998; 273: 5235-5242Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and it is not fully understood how cubilin interacts with the apical lipid bilayer membrane to expose its ligand binding sites to
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