Cbl Complex Research Articles

Binding of aquocobalamin to bovine casein and its peptides via coordination to histidine residues

Vitamin B12 (cobalamin, Cbl) is an essential nutrient of bovine milk, bound to the casein fraction and the Cbl-specific protein transcobalamin at a ratio of 50:50. This study aimed to elucidate the mechanism of interaction between Cbl and the caseins. It was found that the isolated caseins bind aquocobalamin (HOCbl) via histidine-[Co3+]Cbl coordination. The casein–Cbl complex slowly dissociated in the presence of KCN due to formation of CNCbl. The “active” His groups (5.7 mm measured in 41.6 mg mL−1 casein) accumulate ≤ 3 mm HOCbl at a high rate (t½ ≈ 10 min, 20 °C) and with a high affinity (Kd = 0.1 mm). Low pH hinders the binding, but does not accelerate a very slow dissociation (t½ ≈ 8 h). Increased temperature and/or the presence of the specific Cbl-binding proteins accelerate the dissociation. The consequences of casein–Cbl interactions for the intestinal uptake of Cbl remain unclear.

Advanced glycation end products overload might explain intracellular cobalamin deficiency in renal dysfunction, diabetes and aging

Advanced glycation end products (AGEs) contribute to aging. Cobalamin (Cbl) is required for cell growth and functions, and its deficiency causes serious complications. Diabetics and renal patients show high concentrations of Cbl, but metabolic evidence of Cbl deficiency that is reversible after Cbl treatment. Cbl might be sequestered in blood and cannot be delivered to the cell. Megalin mediates the uptake of transcobalamin–Cbl complex into the proximal tubule cells. Megalin is involved in the uptake and degradation of AGEs. In aging, diabetes or renal dysfunction, AGEs might overload megalin thus lowering Cbl uptake. Transcobalamin–Cbl might retain in blood. Shedding of megalin and transcobalamin receptor under glycation conditions is also a possible mechanism of this phenomenon.

Abi-1 forms an epidermal growth factor-inducible complex with Cbl: Role in receptor endocytosis

The Abl-interactor (Abi) proteins are involved in the regulation of actin polymerization and have recently been shown to modulate epidermal growth factor receptor (EGFR) endocytosis. Here we describe the identification of a novel complex between Abi-1 and the Cbl ubiquitin ligase that is induced by stimulation with EGF. Notably, an Abi-1 mutant lacking the SH3 domain (ΔSH3) fails to interact with Cbl and inhibits EGFR internalization. We show that expression of the Abi-1ΔSH3 mutant inhibits Cbl accumulation at the plasma membrane after EGF treatment. We have previously shown that the oncogenic Abl tyrosine kinase inhibits EGFR internalization. Here we report that the oncogenic Abl kinase disrupts the EGF-inducible Abi-1/Cbl complex, highlighting the importance of Abl kinases and downstream effectors in the regulation of EGFR internalization. Thus, our work reveals a new role for oncogenic Abl tyrosine kinases in the regulation of the Abi-1/Cbl protein complex and uncovers a role for the Abi-1/Cbl complex in the regulation of EGFR endocytosis.

Abl Tyrosine Kinase Regulates Endocytosis of the Epidermal Growth Factor Receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.

New insights into the pathophysiology of cobalamin deficiency

Cobalamin-deficient (Cbl-D) central neuropathy in the rat is associated with a locally increased expression of neurotoxic tumour necrosis factor-alpha (TNF-alpha) and a locally decreased expression of neurotrophic epidermal growth factor (EGF). These recent findings suggest that cobalamin oppositely regulates the expression of TNF-alpha and EGF, and raise the possibility that these effects might be independent of its coenzyme function. Furthermore, adult Cbl-D patients have high levels of TNF-alpha and low levels of EGF in the serum and cerebrospinal fluid. Serum levels of TNF-alpha and EGF of cobalamin-treated patients normalize concomitantly with haematological disease remission. These observations suggest that cobalamin deficiency induces an imbalance in TNF-alpha and EGF levels in biological fluids that might have a role in the pathogenesis of the damage caused by pernicious anaemia.

Distinct Turnover of Alternatively Spliced Isoforms of the RET Kinase Receptor Mediated by Differential Recruitment of the Cbl Ubiquitin Ligase

Alternative splicing of transcripts encoding the RET kinase receptor leads to isoforms differing in their cytoplasmic tail. Although in vitro studies have demonstrated a higher transforming activity of the long RET isoform (RET51), only the short isoform (RET9) can rescue the effects of a RET null mutation in the enteric nervous system and kidney development. The molecular basis underlying the distinct functions of the two RET isoforms is not understood. Here we demonstrated that activated RET51 associated more strongly with the ubiquitin ligase Cbl than did RET9, leading to increased ubiquitylation and faster turnover of RET51. The association of Cbl with RET was indirect and was mediated through Grb2. A constitutive complex of Grb2 and Cbl could be recruited to both receptor isoforms via docking of Shc to phosphorylated Tyr-1062 in RET. A mutant Shc protein unable to recruit the Grb2.Cbl complex decreased the turnover and prolonged the half-life of RET9, thus ascribing a previously unknown negative role to the Shc adaptor molecule. In addition, phosphorylation of Tyr-1096, which is present in RET51 but absent in RET9, endowed the longer isoform with a second route to recruit the Grb2.Cbl complex. These findings establish a mechanism for the differential down-regulation of RET9 and RET51 signaling that could explain the apparently paradoxical activities of these two RET isoforms. More generally, these results illustrate how alternative splicing can regulate the half-life and function of a growth factor receptor.

Composite Organization of the Cobalamin Binding and Cubilin Recognition Sites of Intrinsic Factor

Intrinsic factor (IF(50)) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Experiments on binding of Cbl to IF(30), IF(20), and IF(50) revealed comparable association rate constants (k(+)(Cbl) = 4 x 10(6), 14 x 10(6), and 26 x 10(6) M(-1) s(-1), respectively), but the equilibrium dissociation constants were essentially different (K(Cbl) = 200 microM, 0.2 microM, and <or=1 pM, respectively). The smaller fragment, IF(20), had unexpectedly high affinity for Cbl; however, efficient retention of the ligand required the presence of both fragments. Detailed schemes of the interaction of Cbl with IF(50) and with IF(30) and IF(20) are presented, where the sequential attachment of Cbl to the IF(20) and IF(30) domains plays the key role in recognition and retention of the ligand. Each isolated fragment of IF was tested for the binding to the specific receptor cubilin in the presence or absence of Cbl. Neither apo nor holo forms of IF(20) and IF(30) were recognized by the receptor. When two fragments were mixed and incubated with Cbl, they associated into a stable complex, IF(30+20).Cbl, which bound to cubilin as well as the noncleaved IF(50).Cbl complex. We suggest that formation of the cubilin recognition site on IF is caused by assembly of two distant domains, which allows the saturated protein to be recognized by the receptor. The obtained parameters for ligand and receptor binding indicate that both full-length IF(50) and the fragments may be involved in Cbl assimilation.

The sorbin homology domain: a motif for the targeting of proteins to lipid rafts.

On phosphorylation of Cbl, the c-Cbl-associated protein (CAP)/Cbl complex dissociates from the insulin receptor and translocates to a lipid raft membrane fraction to form a ternary complex with flotillin. Deletion analyses of the CAP gene identified a 115-aa region responsible for flotillin binding. This region is homologous to the peptide sorbin and is referred to as the sorbin homology (SoHo) domain. This domain is present in two other proteins, vinexin and ArgBP2. Vinexin also interacted with flotillin, and deletion of its SoHo domain similarly blocked flotillin binding. The overexpression of a CAP mutant in which the SoHo domain had been deleted (CAPDeltaSoHo) prevented the translocation of Cbl to lipid rafts and subsequently blocked the recruitment of CrkII and C3G. Moreover, overexpression of CAPDeltaSoHo prevented the stimulation of glucose transport and GLUT4 translocation by insulin. These results suggest a mechanism for localization of signaling proteins to the lipid raft that mediates the compartmentalization of crucial signal transduction pathways.

CAP defines a second signalling pathway required for insulin-stimulated glucose transport.

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl proto-oncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Assimilation of [57Co]-labeled cobalamin in human fetal gastrointestinal xenografts into nude mice.

Cobalamin (Cbl) and its Cbl-binding proteins are present in amniotic fluid. Because amniotic fluid is swallowed by the embryo-fetus, we studied the ability of Cbl to be transported and metabolized across the embryo-fetal digestive tract. Human embryonic stomachs and intestines were transplanted into nude mice. The basal secretion of Cbl-binding proteins was studied by gel filtration of the graft juices. Intrinsic factor (IF) was looked for in gastric mucosa by immunohistochemistry. The uptake of [57Co]-labeled Cbl by the intestinal graft was studied by Schilling tests and HPLC. IF, haptocorrin, and a transcobalamin-like protein were detected in gastric juice, with concentration ranges of 5.0-26.4, 1.9-27.1, and 5.2-12.6 pmol/mL, respectively. The IF [57Co]Cbl complex had a single isoprotein with a pI at 5.6, which was maintained after incubation with neuraminidase. Urine excretion percentages (Schilling tests) ranged from 5.5 to 21.2% and from 0.3 to 1.6% when cyano-[57Co]Cbl-IF or cyano-[57Co]Cbl, respectively, was instilled in intestinal grafts. Chloroquine reduced significantly the percentage of excreted [57Co]Cbl. The [57Co]Cbl was mainly excreted as cyano-[57Co]Cbl in urines, showing a low coenzyme conversion. In conclusion, IF is secreted by the nonstimulated embryonic stomach and lacks sialic acid. Cbl binds to it and is subsequently transported across the xenografted embryo-fetal intestine. This suggests that amniotic fluid may contribute to Cbl delivery to the embryo-fetus.

Identification of rat yolk sac target protein of teratogenic antibodies, gp280, as intrinsic factor-cobalamin receptor.

Previous studies in the rat have shown that antibodies to gp280, a protein > 200 kD and closely associated with the early endocytic system can induce fetal malformations. Although gp280 is thought to act as a receptor, its ligand(s) is not known. In the current study, we report that purified gp280 from rat kidney, like the intrinsic factor-Cobalamin receptor (IFCR), binds to the intrinsic factor-cobalamin (IFCbl) complex with an association constant of 0.3 x 10(9) M-1 and mediates its internalization. Furthermore, antibodies raised to purified gp280 and IFCR inhibited the binding of IF-[57Co]Cbl complex to intestinal, renal, and yolk sac apical membranes and revealed a single identically sized protein on immunoblotting of the renal membranes. Both antibodies precipitated a single radiolabeled protein > 200 kD from cellular extract from [35S]methionine-labeled yolk sac epithelial cells, and antibody to gp280 inhibited the uptake and internalization of 125IF-Cbl. Immunoelectron microscopy using the two antibodies revealed that in the kidney, both proteins were colocalized. These observations suggest that IF-Cbl complex is a ligand for gp280 and that gp280 and IFCR are identical proteins.

Functional expression of transcoabalamin II cDNA In [formula omitted] oocytes

The products of in vitro transcription of human transcobalamin II (TC II) cDNA when microinjected into Xenopus laevis oocytes yielded a single secretory protein of 43 kDa. The mobility of the 43 kDa band did not change following digestion with peptide N-glycosidase F. [ 57Co]Cbl bound to the medium was immunoprecipitated with anti-serum to human TC II, but not to other Cbl binders. In addition, the [ 57Co]Cbl complex also bound to placental microsomes. These results suggest that TC II mRNA transcribed encodes TC II which contains both the Cbl and receptor binding domains. Furthermore, Xenopus oocytes can be used as a screening system to define structural elements important in TC II's secretion and binding reactions.

Cobalamin release from intrinsic factor and transfer to transcobalamin II within the rat enterocyte

To ascertain the mechanism of release of cobalamin (Cbl) from intrinsic factor (IF) and subsequent formation of transcobalamin II (TC-II)-Cbl complex, we studied the intracellular distribution of 57Co-labeled Cbl after its uptake in suckling and adult rats. The amount of Cbl bound to IF, to the IF-Cbl receptor via IF, and to TC-II was determined by immunoprecipitation with monospecific antisera raised to these proteins. IF-Cbl receptor activity was found to be very low in suckling rats up to 12 days after birth. Oral administration of leupeptin in amounts known to alter protein turnover had no effect on the release of Cbl from IF nor did it inhibit the formation of the TC-II-Cbl complex in either adult or suckling animals. However, oral administration of chloroquine resulted in a transient increase in the intestinal concentration of Cbl in both adult and suckling rats and in total inhibition of Cbl released from IF in adults rats. Chloroquine prevented completely the transfer of Cbl to TC-II in adult rats and inhibited the transfer by 50% in suckling rats. These data demonstrate that in adult mucosa utilizing receptor-mediated endocytosis, Cbl is transferred from IF to TC-II. This transfer does not require the IF-Cbl receptor, as it occurs in suckling rats. Finally, transfer of Cbl to TC-II is decreased by a drug that alters vesicular pH. Because Cbl can be released at acid pH from IF, it is proposed that release of Cbl from IF and its transfer to TC-II occurs in an acidic vesicle.

3] Isolation of native and proteolytically derived ileal receptor for intrinsic factor—cobalamin

This chapter describes the assay method, purification, and isolation of native and proteolytically derived ileal receptor for intrinsic factor–cobalamin. Receptor activity during the purification was assessed by measuring the binding of the hog IF-[ 57 Co]Cbl complex made with purified hog intrinsic factor (IF). Hog IF can be replaced by human IF. However the important consideration is that IF preparations should be free of any possible contamination of other Cbl-binding proteins (nonIF). The supernatant fraction containing unbound IF-[ 57 Co]Cbl and the pellet containing receptor bound IF-[ 57 Co]Cbl are assayed for radioactivity. Receptor specific binding is calculated as the difference between the binding observed in the presence of CaCl 2 and in the absence of calcium with EDTA. The most important step in the purification is the immunoaffinity chromatography using rabbit anti-hog intrinsic factor bound to Sepharose 4B. For the purification of the dog receptor attempts to use either intrinsic factor alone or the intrinsic factor-cobalamin complex as an affinity absorbent resulted in very poor binding of the receptor compared to rabbit anti-hog intrinsic factor antiserum. The advantage of this method is the recycling of both the intrinsic factor-cobalamin complex and the affinity ligand. After elution of the receptor the column can be washed with 0.2 M glycine-HCl buffer, pH 3.0, to remove the intrinsic factor-cobalamin complex which was bound to the antibody. The eluate is neutralized to pH 7 and can be reused after assessing the amount of complex present.

Intestinal uptake and release of cobalamin complexed with rat intrinsic factor.

The mechanism of uptake of intrinsic factor (IF) and cobalamin (Cbl) by enterocytes and their subsequent fate have been uncertain. To examine this problem double-labeled IF X Cbl was added to small intestinal organ cultures. When 125I-IF X [57Co]Cbl was added to rabbit ileal explants, binding and internalization increased linearly for 24 h. After an 18-h chase with nonlabeled IF X Cbl, no 125I-IF returned to the cell surface. An amount of 35-45% of the internalized Cbl was found free, not bound to IF or any other protein. About 60% of both internalized ligands was bound to membranes but by a non-Ca2+-dependent bond, suggesting binding to a protein other than the brush-border receptor. Cobalamin was released from IF at pH 5.0 to the same degree (30%) as free Cbl was found inside the cell (35-45%). Neither pancreatic proteases nor ileal homogenates effected release of Cbl from IF. When cathepsins were added, the Cbl released was no greater than could be attributed to pH 5.0 alone. Chloroquine added to tissue explants did not alter the percentage of free intracellular Cbl. From these results we suggest that IF X Cbl is internalized and detached from the receptor within the enterocyte. The mechanism of release is not known but seems to require an acid pH (5.0). The Cbl is released in the mucosa, perhaps when the IF X Cbl complex enters a nonlysosomal cellular compartment with an acidic environment. There is no substantial recycling of IF to the brush-border membrane.

Characterization of cobalamin receptor sites in brush-border plasma membranes of the tapeworm Spirometra mansonoides.

The interaction of receptor sites in microtriches (brush-border) membranes isolated from the sparganum (larva) of the cestode (tapeworm) Spirometra mansonoides with [57Co]cyanocobalamin (CN-[57Co]Cbl) has been characterized on the basis of the following criteria: time dependence, reversibility, high-affinity binding, finite binding capacity, and ligand specificity. The association of CN-[57Co]Cbl to isolated microtriches membranes at 22 degrees C reached equilibrium in approximately 90 min. The initial rate of association follows second order kinetics with an association rate constant (ka) of 1.1 X 10(6) M-1 S-1. In the presence of an excess of unlabeled CN-Cbl, the receptor CN-[57Co]Cbl complex dissociated according to first order kinetics with a dissociation constant (kd) of 4.4 X 10(-4) S-1. An equilibrium dissociation constant (KD) of 0.4 nM was calculated on the basis of rate constants (KD = kd/ka). Scatchard analysis of equilibrium-binding data revealed a single class of high affinity binding sites with a KD of 0.3 nM and a maximum binding capacity for CN-Cbl of 221 fmol/mg of membrane protein. The stereo-specificity of CN-Cbl binding to receptor sites in isolated microtriches membranes was determined using various Cbl analogs. Receptor-site binding affinity was lowered by the following modifications: deamination of the b-, d-, and e-propionamide side chains to the corresponding monocarboxylate derivatives; modification of the B pyrrole ring to lactam or lactone; substitution of the benzimidazole moiety by a purine; modification of the benzimidazole; or deletion of the entire nucleotide moiety. On the basis of the action of trypsin and pronase, this CN-Cbl receptor may contain a relatively exposed membrane protein. The physiological role of this CN-Cbl receptor in the binding, internalization, and metabolism of Cbl is discussed in relation to cestodes and their mammalian hosts.