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 designed to evaluate the functional role of TSHR carbohydrates in detail. Because our previous mutagenesis study by Asn to Gln substitutions suggested the critical role of the first and third glycosylation sites (amino acids 77 and 113) for expression of the functional TSHR, we first constructed TSHR mutants 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 glycosylation site were found to be intact. These data indicate that our previous data appear to result from amino acid substitution itself, not from disruption of glycosylation. The next series of the mutants was therefore constructed to identify at least how many glycosylation sites are necessary. Neither TSH binding nor cAMP response was detected in TSHR mutants with three glycosylation sites. However, the mutants with four glycosylation sites were fully functional in terms of TSH binding and cAMP production, although the expression levels were 30 to 40% of that in wild-type TSHR. Finally, Western blot revealed that all six glycosylation sites are actually glycosylated. These data indicate that 1) TSHR ectodomain contains six N-linked carbohydrates, and 2) glycosylation of at least four sites appears necessary for expression of the functional TSHR.
Nagayama
Y
Y
Department of Pharmacology 1, Nagasaki University School of Medicine, Nagasaki, Japan. nagayama@net.nagasaki-u.ac.jp
Nishihara
E
E
Namba
H
H
Yamashita
S
S
Niwa
M
M
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D017510:Protein Folding; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship
D011989:Receptors, Thyrotropin; D001216:Asparagine