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. The LHR1 is a glycoprotein present in the cell membrane of gonadal cells, with six potential N-linked glycosylation sites ( Fig. 1) and N-linked carbohydrate (CHO) chains of the complex type (1-3). A functional role for N-linked carbohydrates in high affinity hormone binding has not yet been established, and is a subject of some controversy. Conflicting reports on the effects of deglycosylation (2, 4) and site-directed mutagenesis (5, 6) of the mature holoreceptor on hormone binding activity may be a function of the original receptor state. N-Linked carbohydrate chains of the rat ovarian LH receptor have previously been shown to be essential for high affinity hormone binding by a number of different methods, including site-directed mutagenesis of the Asn of the putative glycosylation sites (5), tunicamycin treatment (7), and enzymatic deglycosylation of solubilized receptors (2). These procedures have not resolved the question of whether the reported carbohydrate requirement of the receptor is based on a direct interaction with the hormone or is related to the conformation of the ligand binding site.In order to study the importance of post-translational glycosylation on LHR binding activity, we have evaluated both the membrane-bound LHR holoreceptor (form A) (3) and its highaffinity hormone-binding splice variant, form B, which lacks the transmembrane and cytoplasmic domains (8). These receptors and their mutated forms were expressed in Baculovirusinfected insect cells and in the mammalian COS-1 cells. It is noteworthy that glycoproteins in insect cells h...

No abstract