Mr 46,000 mannose 6-phosphate receptor. The role of histidine and arginine residues for binding of ligand.
The chemical modification of histidine and arginine residues results in a loss of binding of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) to a phosphomannan affinity matrix (Stein, M., Meyer, J. E., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 927-936). Reversal of the modification or presence of mannose 6-phosphate during the modification partially restores or protects the binding activity, indicating that histidine and arginine residues contribute to the mannose 6-phosphate binding site. The 5 histidine and 8 arginine residues within the luminal domain of MPR 46, which contains the ligand binding site, were exchanged by site-directed mutagenesis. Only the conservative replacement of His-131 and Arg-137 by serine and lysine, respectively, results in a loss of binding activity without affecting other properties of the receptor such as the presence of intramolecular disulfide bonds, immunoreactivity, processing of N-linked oligosaccharides, formation of dimers, intracellular distribution, and surface expression. Conservative replacement of other histidine and arginine residues did not affect the binding activity. Nonconservative replacement of several arginine residues reduced binding activity and immunoreactivity, indicating that the loss of a positive charge at these positions alters the folding of MPR 46. We conclude from these results that His-131 and Arg-137 are essential for binding of ligands by MPR 46.
Wendland
M
M
Universitat Göttingen, Abteilung Biochemie II, Federal Republic of Germany.
Waheed
A
A
von Figura
K
K
Pohlmann
R
R
D001120:Arginine / Q000235:genetics*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000235:genetics*; D007700:Kinetics; D008024:Ligands; D008360:Mannosephosphates / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*
D008024:Ligands; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D006639:Histidine; D001120:Arginine