Membrane expression and interactions of human transcobalamin II receptor.
Antiserum raised to purified 62-kDa human placental transcobalamin II receptor (TC II-R) has been used to study its synthesis and membrane expression. The antiserum immunoprecipitated a 45-kDa protein from the cell-free translation using human kidney mRNA and recognized a single 124-kDa band on immunoblotting of placental and other human tissue membranes, and quantitation of the blots revealed high levels of TC II-R expression in the human kidney followed by placenta, intestine, and liver. Triton X-100 extraction of placental membranes resulted in the complete (100%) solubilization of the receptor, and immunoblotting of the Triton X-100-soluble fraction revealed a single band of 62 kDa. Lipid extraction of placental membranes with a mixture of chloroformmethanol (2:1) followed by immunoblotting revealed a single band of molecular mass 62 kDa. The molecular mass of the pure Triton X-100-bound receptor increased on SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its insertion in liposomes prepared using egg phosphatidylcholine and cholesterol. Chemical cross-linking of native membrane-or lipid vesicle-bound TC II-R or detergent-soluble extracts of the membrane with 125I-TC II-cobalamin revealed that both the 124- and 62-kDa forms of the receptor were active in ligand binding. Based on these results we suggest that TC II-R is synthesized as a single polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is expressed in plasma membranes as a noncovalent dimer of 124 kDa. The dimerization of TC II-R in the plasma membranes is due to its interactions with annular lipids.
Bose
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S
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, USA.
Seetharam
S
S
Seetharam
B
B
D001704:Biopolymers; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D008563:Membrane Lipids; D011956:Receptors, Cell Surface; C028176:transcobalamin receptor