The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.
The extracytoplasmic region of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) consists of 15 homologous repeating domains, each of which is approximately 147 residues in length. The receptor contains two high affinity mannose 6-phosphate (Man-6-P) binding sites and our recent studies (Westlund, B., Dahms, N. M., and Kornfeld, S. (1991) J. Biol. Chem. 266, 23233-23239) have localized these two binding sites to domains 1-3 and 7-11. To further define the location of the Man-6-P binding sites and to determine the role of specific arginine residues in Man-6-P binding, site-directed mutagenesis was utilized to create truncated soluble forms of the M6P/IGF-II receptor in conjunction with either conservative (Lys) or nonconservative (Ala) replacement of arginine residues. These mutants were expressed transiently in COS-1 cells and assayed for their ability to bind phosphomannosyl residues by affinity chromatography. Analysis of the ligand binding activity of carboxyl-terminal truncated forms of the receptor's extracytoplasmic region demonstrated that the second Man-6-P binding site is contained within domains 7-9. Substitution of Arg435 in domain 3 of the amino-terminal binding site and Arg1334 in domain 9 of the second binding site results in a dramatic loss of ligand binding activity. However, substitutions at positions 435 and/or 1334 did not affect the secretion, glycosylation, or immunoreactivity of these truncated proteins. Taken together, these results indicate that Arg435 and Arg1334 are essential components of the M6P/IGF-II receptor's high affinity Man-6-P binding sites.
Dahms
N M
NM
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Rose
P A
PA
Molkentin
J D
JD
Zhang
Y
Y
Brzycki
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D004247:DNA; D008360:Mannosephosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D004247:DNA; D001120:Arginine