title: Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin.
abstract: The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of the receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (Ka=10 nM(-1)) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular region (aa 32-229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95-141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (Ka = 19-24 nM(-1)). The two LDLR-A domains (aa 54-89 and 132-167) with the negatively charged acidic residues involved in Ca(2+) binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPLGLL motif and the PDZ binding motifs (QERL/KESL) appear to be involved in initiating and completing the process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency.
authors.affiliation: School of Graduate Studies, State University of New York Downstate Medical Center, Brooklyn, New York 11203, USA.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Jiang
authors.forename: Yasumi
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Sequeira
authors.forename: Edward V
authors.initials: EV
authors.lastname: Quadros
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D054731:PDZ Domains / Q000235:genetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods; D011956:Receptors, Cell Surface; D011973:Receptors, LDL / Q000378:metabolism; D017384:Sequence Deletion; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; C538894:CD320 protein, human; C573412:LDLR protein, human; D008024:Ligands; D011956:Receptors, Cell Surface; D011973:Receptors, LDL; D014155:Transcobalamins; D049452:Green Fluorescent Proteins; D014805:Vitamin B 12
keywords: Hek293 cells; LDLR-A; Qdots; calcium-binding domain; cytoplasmic domain; vitamin B12