Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor.
Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007784:Lactoperoxidase / Q000378:metabolism; D008081:Liposomes; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D017527:Receptor, IGF Type 2; D014357:Trypsin / Q000378:metabolism
D002352:Carrier Proteins; D008081:Liposomes; D010446:Peptide Fragments; D017527:Receptor, IGF Type 2; D007784:Lactoperoxidase; D002918:Chymotrypsin; D014357:Trypsin