Identification of the sites of asparagine-linked glycosylation on the human thyrotropin receptor and studies on their role in receptor function and expression

The amino-terminal ectodomain of human thyrotropin receptor (TSHR) contains six potential N-linked glycosylation sites (N-Xaa-S/T). This study was des igned to evaluate the functional role of TSHR carbohydrates in detail. Beca use our previous mutagenesis study by Asn to Gln substitutions suggested th e critical role of the first and third glycosylation sites (amino acids 77 and 113) for expression of the functional TSHR, we first constructed TSHR m utants having these two glycosylation sites to elucidate whether these two sites are sufficient for TSHR function and expression; this mutant however proved to be nonfunctional. Also the expression levels and function of TSHR mutants with a Ser/Thr to Ala substitution at the first or third glycosyla tion site were found to be intact. These data indicate that our previous da ta appear to result from amino acid substitution itself, not from disruptio n of glycosylation. The next series of the mutants was therefore constructe d to identify at least how many glycosylation sites are necessary. Neither TSH binding nor cAMP response was detected in TSHR mutants with three glyco sylation sites. However, the mutants with four glycosylation sites were ful ly functional in terms of TSH binding and cAMP production, although the exp ression levels were 30 to 40% of that in wild-type TSHR. Finally, Western b lot revealed that all six glycosylation sites are actually glycosylated. Th ese data indicate that 1) TSHR ectodomain contains six N-linked carbohydrat es, and 2) glycosylation of at least four sites appears necessary for expre ssion of the functional TSHR.