The oligomeric state of 46-kDa mannose 6-phosphate receptor does not change upon intracellular recycling and binding of ligands.
46-kDa mannose-6-phosphate receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa mannose 6-phosphate receptor is regulated by the presence of ligands, pH and receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47-54). To find out whether the intracellular recycling of 46-kDa mannose 6-phosphate receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa mannose 6-phosphate receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20% of total endogenous 46-kDa mannose 6-phosphate receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa mannose 6-phosphate receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa mannose 6-phosphate receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46-kDa mannose 6-phosphate receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa mannose 6-phosphate receptor in situ, our data suggest that it represents the main physiologically active form of the receptor, and therefore present indirect evidence that binding of ligands to 46-kDa mannose 6-phosphate receptor is probably regulated by conformational changes of receptor or ligand rather than by changes in the quaternary structure.
Punnonen
E L
EL
Universität Göttingen, Institut für Biochemie II, Germany.
Wilke
T
T
von Figura
K
K
Hille-Rehfeld
A
A
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003432:Cross-Linking Reagents; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D015388:Organelles / Q000378:metabolism; D011487:Protein Conformation; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008024:Ligands; D017527:Receptor, IGF Type 2