Mutational analysis of disulfide bridges in the Mr 46,000 mannose 6-phosphate receptor. Localization and role for ligand binding.
Formation of intramolecular disulfide bonds is a key step in the early maturation of newly synthesized Mr 46,000 mannose 6-phosphate receptors to acquire ligand-binding activity (Hille, A., Waheed, A., and von Figura, K. (1990) J. Cell Biol. 110, 963-972). The luminal domain of the receptor, which carries the ligand-binding site, contains 6 cysteine residues. We have analyzed the function of individual cysteine residues for the ligand-binding conformation by exchanging cysteine for glycine. In each case, the replacement of cysteine resulted in a complete loss of binding activity, indicating that all 6 luminal cysteine residues are required for the ligand-binding conformation. The cysteine mutants displayed a greatly reduced immunoreactivity, decreased stability, and a blocked or delayed transport to the trans Golgi. The glycosylation pattern allowed the distinguishing of three phenotypes, each of which was represented by one pair of cysteine mutants. Based on the assumption that replacement of either of the 2 cysteine residues forming a disulfide bond results in an identical phenotype, we postulate that disulfide bonds are formed between Cys-32 and Cys-78 and between Cys-132 and Cys-167, as well as between Cys-145 and Cys-179. This assumption was supported by the observation that the simultaneous exchange of the 2 cysteine residues of a putative pair resulted in the same phenotypes as the single exchange of either of the 2 cysteine residues.
Wendland
M
M
Universität Göttingen, Abteilung Biochemie II, Republic of Germany.
von Figura
K
K
Pohlmann
R
R
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D008360:Mannosephosphates / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004220:Disulfides; D008024:Ligands; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface