Figure 1.
Schematic representation of the TSHR mutants (A623V and I691F).
The mutants with A623V and I691F desciribed in this article. A623V and I691F locte in third intracellular loop and cytoplasmic tail region of TSHR respectively.
Figure 2.
Characterization of chimeric constructs of TSHR and fluorescent reporters.
(A) COS-7 cells transfected with GFP chimeras of wild-type or TSHR mutants and visualized by fluorescence microscopy. Wild-type TSH receptor is expressed at the plasma membrane. Mutant A623V and I691F receptors are poorly expressed at the plasma membrane and are retained in intracellular regions; and (B) Western blot analysis of different GFP chimeras using an anti-human TSHR antibody (H-115) antibody. The band consistent with the molecular weights of receptor (∼90 kDa, TSHR + GFP) forms were present.
Figure 3.
Cell surface expression determined by Flow cytometry analysis.
(A) COS-7 cells were assayed after transfection with pEGFP-N1-vector, wild-type TSHR, mutants A623V and I691F. Fluorescence intensity is expressed in arbitrary units as a function of cell number plotted on a logarithmic scale. Representative mutants are shown. (B) Expression level of the pEGFP-N1, wild-type TSHR and mutants with A623V and I691F. Data are expressed as percentage expression of wild-type TSHR (set at 100%) and are presented as means ± S.E.M. of three independent experiments, each carried out in duplicate. *p<0.05; **p<0.001.
Table 1.
Functional characterization of TSHR mutants.
Figure 4.
Basal and TSH-stimulated cAMP accumulation.
cAMP accumulation assays were performed with transiently transfected COS-7 cells with pEGFP-N1-vector, wild-type TSHR and mutants with A623V and I691F. Forty-eight hours after transfection, COS-7 cells were incubated in 10 or 100 mU/ml bTSH. cAMP levels were determined as described in materials and methods. Data are expressed as relative to wild-type basal and given as means ± S.E.M. of three independent experiments, each carried out in triplicate. *p<0.05; **p<0.001.
Figure 5.
Basal and TSH-stimulated IP accumulation.
COS-7 cells were transfeted with pEGFP-N1-vector, wild-type TSHR and mutants with A623V and I691F. Twenty-fourt hours after transfection, cells were incubated in 10 or 100 mU/ml bTSH. IP accumulation was determined as described in materials and methods. The basal IP production of the wild-type TSHR was set at 1 and all other data are expressed as fold of the basal IP production of the wild-type TSHR. Data are presented as means ± S.E.M. of two independent experiments, each carried out in triplicate. *p<0.05; **p<0.001.
Figure 6.
The alignment of amino acids in the cytoplasmic tail segment.
Alignment of the cytoplasmic tail segment of the TSHR with different species, Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus familiaris, Felis catus, Gallus gallus, Dicentrarchus labrax, Solea senegalensis, Sus scrofa, Equus caballus, Chlorocebus aethiops, Macaca mulatta. The sequence of the cytoplasmic tail region of the TSHR between residues 688 and 695 including Ile691 (*) is highly conserved in various species.