Effects of mutations involving cysteine residues distal to the S281HCC motif at the C-terminus on the functional characteristics of a truncated ectodomain-only thyrotropin receptor anchored on glycosylphosphatidyl-inositol.
BACKGROUND
Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR.


METHODS
We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression.


RESULTS
In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity.


CONCLUSIONS
From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.
Ho
Su-Chin
SC
SingHealth Research, Singapore Health Service, Singapore, Republic of Singapore. ho.su.chin@sgh.com.sg
Goh
Sui-Sin
SS
Li
Sun
S
Khoo
Daphne Hsu-Chin
DH
Paterson
Malcolm
M
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000235:genetics*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D003545:Cysteine