Alanine mutagenesis and functional characterization of amino acids in the ICL1 and transitions to the helices 1 and 2.

COS-7 cells were transfected with wt TSHR or various mutant TSHRs. The vector pcDNA3.1(−) / hygromycin was used as a control. The TSHR is characterized by an elevated cAMP level compared to the control vector alone [76]. Therefore, cAMP accumulation is expressed relative to wt TSHR basal level. TSH-mediated levels of cAMP and IP accumulation were determined after treatment of cells with 100 mU/ml bTSH. Expression of wt and mutant TSHRs were quantified on a FACS flow cytometer. Data are given as mean ± standard deviation (SD) of at least three independent experiments (n = 3), each carried out in duplicate. Constitutive activity by linear regression analyses was determined for mutant L439A. aPbP = 0.001 to 0.01, cP = 0.01 to 0.05.