Functional glycosylation sites of the rat luteinizing hormone receptor required for ligand binding.
The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.
Zhang
R
R
Section of Molecular Endocrinology, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Cai
H
H
Fatima
N
N
Buczko
E
E
Dufau
M L
ML
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000378:metabolism; D005260:Female; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D010053:Ovary / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011974:Receptors, LH / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D006063:Chorionic Gonadotropin; D008024:Ligands; D009844:Oligosaccharides; D011974:Receptors, LH; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases