FUNCTIONAL GLYCOSYLATION SITES OF THE RAT LUTEINIZING-HORMONE RECEPTOR REQUIRED FOR LIGAND-BINDING

The contribution of N-linked glycosylation to the ligand binding activ ity of the rat luteinizing hormone receptor (LHR) was studied in wild- type and mutant LHR expressed in mammalian (COS1) cells and overexpres sed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane do main were equivalent in both cell lines. Tunicamycin-treated transfect ed Sf9 cells expressed a carbohydrate-free LH receptor that lacked hor mone binding activity. Functional carbohydrate chains essential for bi nding activity were localized to glycosylation sites at Asn-173 and As n-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 bind ing. LHR expressed in insect cells contained only high mannose carbohy drate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl cha ins indicated that only the proximal N-acetylglucosamine residue, whic h is common to high mannose and complex carbohydrate forms, is necessa ry for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolec ular folding of the nascent receptor rather than in its interaction wi th the hormone.