|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1997 : ¥1,900,000 (Direct Cost : ¥1,900,000)
1. Glvcosylation of thyrotropin (TSH) receptor - TSH receptor ectodomain is heavily glycosylated with N-linked carbohydrates. In immunoprecipitation, two types of TSH receptor can be dctected - a precursor form with high mannose type carbohydrates and a mature protein with complex type carbohydrates. Using a glycosylation inhibitor tunicamycin and a series of mutant CHO cells (CHO-Lec) defective in the different steps of glycosylation processing, we found that acquisition of carbohydrates is essential for proper folding of TSH receptor in endoplasmic reticulum and its processing is involved in cell surface targeting in Golgi apparatus.
2. Acylation of TSH receptor - We demonstrate a cysteine residue at amino acid 699 of the C-terminal cytoplasmic tail of the receptor is palmitoylated. Stuides with a mutant TSH receptor lacking palmitoylationreveal that palmitoylation plays a pivotal role in intracellular trafficking of the receptor, but not in high affinity TSH binding, Gs coupling, homologous desensitization or internalization.
3. Subunit structure of TSH receptor - TSH receptor is well known to cleave into two subunits, A and B.However, we found that there may be two cleavage sites, releasing not only two subunits but also a small peptide (-7-8 kDa). Even extensive mutagenesis studies around possible cleavage sites failed to identify the exact cleavage site, suggesting proteolytic enzyme(s) may recognize the specific three dimensional structure, not specific amino acids, of the receptor.