TSH binding to TSHR with four and five charged residues mutated to alanine.

TSHR IV (K244A, E247A, K250A, R255A), TSHR V (K244A, E247A, K250A, E251A, R255A) and the wild-type TSHR stably expressed in CHO cell monolayers were incubated for 4 h at room temperature in buffer containing 125I-TSH supplemented with the indicated concentrations of unlabeled TSH (Methods). Data from four separate experiments are expressed as the mean ± S.E.M. of radioactivity bound as a percentage of total radioactivity added per well (typically ∼30,000 cpm).*p = 0.004, Student's t-test.