Posttranslational modifications of the lutropin receptor: Mass spectrometric analysis

Our present knowledge of the lutropin (LH/hCG) receptor structure derives f rom deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone ( LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature re ceptor, complexed to human chorionic gonadotropin (hCG), was purified by us ing monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodi es. Determination of the N-terminus showed that 2/3 of protein molecules st arted at Thr24 whereas 1/3 Started at Ala28. All. these molecules bound hCG , suggesting that the most N-terminal region of the receptor does not parti cipate in hormone binding. Six N-glycosylation sites have been predicted fr om the amino acid sequence. One of them (Asn299) was found to be nonglycosy lated in both the precursor and the mature protein. The most heavily glycos ylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor a nd in the mannose-rich precursor, respectively. The presence of some recept or molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone b inding or in the correct folding of the protein. The mature carbohydrate ch ains were homogeneous at position 174, 195, and 313 (absence of Golgi manno sidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were prese nt at sites 99 and 291. The latter, which is highly variable in carbohydrat e chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.