Figure 1.
Schematic representation of the three components of the TSHR.
The leucine-rich repeat domain (LRD) is linked to the serpentine transmembrane domain (TMD) by the hinge region. The crystal structure of the TSHR LRD has been solved [6](Protein Data Base 3G04) and is shown using FirstGlance in Jmol (http://molvis.sdsc.edu/fgij/). The TMD structure can be modeled with reasonable confidence from the crystal structures of other Group A, rhodopsin-like, GPCR members [1]–[4]. The structure of the intervening hinge region is totally unknown. A. Depiction of all three components. TSH binds largely to the LRD with a smaller contribution to the binding site by the hinge region. Figure 1A is a modification of Figure 6 in Ref. [15]. B. Side view of TSHR LRD. Charged residues at the C-terminal base of the LRD are indicated by the yellow halos. C. Inferior aspect of the TSHR LRD.
Figure 2.
Functional effects of mutating charged residues at the C-terminal base of the TSHR LRD, singly and in combination.
Unless indicated otherwise, all mutations were to alanine. CHO cells stably expressing TSHR with the indicated mutations, as well as the wild type TSHR, were incubated for 1 h in the indicated concentrations of TSH and intracellular cAMP measured (Methods). In each panel, the points represents the mean ± range of cAMP values in duplicate wells of cells. Values are expressed as a percentage of the maximal cAMP attained. The TSH effective concentrations required for half-maximal stimulation (EC50) in these representative experiments are indicated by the horizontal dashed line. The mean values and statistical analysis of data from multiple experiments are depicted in Figure 3. Panel A. Single mutations to alanine of charged residues at the base (C-terminus) of the TSHR LRD. For comparison, the previously published effect of an E251K mutation on the cAMP response to TSH stimulation [15] is shown in red. In the experiment shown, with basal cAMP levels of 0.8–1.3 pmoles per well of cells, mean maximum cAMP values attained were 60.5 (wild-type), 58.7 (K244A), 52.2 (E247A), 39.4 (K250A), 49.9 (E251A) and 66.1 (R255A) pmoles per well of cells. Panel B. Double mutations to alanine of charged residues at the C-terminus of the TSHR LRD. In the experiment shown, with basal cAMP levels of 0.5–0.8 pmoles per well of cells, mean maximum cAMP values were 60.5 (wild-type), 52.1 (E251,244A), 51.7 (E251A,K247A), 48.2 (E251A,K250A), and 54.7 (E251A,R255A) pmoles per well of cells. Panel C. Triple mutations to alanine of charged residues at the base of the TSHR LRD. In the experiment shown, with basal cAMP levels of 0.9–1.4 pmoles per well of cells, mean maximum cAMP values were 69.3 (wild-type), 57.9 (E251A,K250A,K244A), 55.2 (E251A,K250A,E247A) and 53.5 (E251A,K250A,R255A) pmoles per well of cells. Panel D. Combination of four (IV) and five (V) mutations to alanine of charged residues at the base of the TSHR LRD. In the experiment shown, with basal cAMP levels of 0.6–1.0 pmoles per well of cells, mean maximum cAMP values were 69.3 (wild-type), 42.9 (K244A,E247A,K250A,R255A), and 33.4 (K244A,E247A,K250A,E251A,R255A) pmoles per well of cells. Panel E. Single mutations of K244, E247, K250 and R255 to residues with an opposite charge. In the experiment shown (representative of three), with basal cAMP levels of 0.7–1.4 pmoles per well of cells, mean maximum cAMP values were 27.3 (wild-type), 31.7 (K244E), 35.8 (E247K), 34.6 (K250E) and 34.5 (R255E) pmoles per well of cells.
Figure 3.
Summary and comparison of the sensitivities to TSH stimulation in all groups with alanine substitutions.
The numbers besides bars indicate the amino acid residues that were mutated to alanine. Each bar represents the mean+S.D. of the EC50s for TSH stimulation of cAMP generation. The number of experiments is indicated in parentheses. Statistical comparison of each groups vs. the wild-type TSHR (wt) was performed by One-way ANOVA.
Figure 4.
Thyroid stimulating monoclonal autoantibody M22 stimulation of TSHR variants.
CHO cells stably expressing the wild-type TSHR and the TSHR with four residues (K244,E247,K250,R255) and five residues (K244,E247,K250,E251,R255) converted to alanine, termed ‘IV’ and ‘V’ respectively, were incubated for 1 h in the indicated concentrations of M22 and intracellular cAMP measured (Methods). Each point represents the mean ± range of cAMP values in duplicate wells of cells. The experiment shown is representative of three separate experiments. The very low responses of TSHR-IV and TSHR-V preclude determination of the M22 EC50's, so absolute cAMP values are shown. The M22 EC50 for the wild-type TSHR is indicated by the horizontal dashed line. In all other experiments in which there was a robust cAMP response, for better visualization of the EC50, cAMP values were normalized to 100% of the maximal value attained.
Table 1.
TSH binding to TSHR with four and five charged residues mutated to alanine.
Figure 5.
Cell surface expression by TSHR mutants determined by flow cytometry.
TSHR mutants were stably expressed in CHO cells. The numbers below each bar indicate the TSHR residue mutated without, for simplicity, the specific amino acid substitution. Each cell line was tested with the three indicated monoclonal antibodies. Geometric mean fluorescence for each is net after subtraction of fluorescence obtained for each mAb with control, untransfected CHO cells (9–10 fluorescence units). Backgrounds with normal IgG controls for each mutant cell line were ∼2–6 fluorescence units. The vertical arrows indicate the low fluorescence observed with mAb CS-17 relative to the other two mAb, all such cell types involving mutation of residue K244 and/or E247.
Figure 6.
TSHR residues affecting monoclonal antibody CS-17 binding do not alter the cAMP response to TSH stimulation.
CHO cells stably expressing TSHR with the indicated mutations, as well as the wild type TSHR, were incubated for 1 h in the indicated concentrations of TSH and intracellular cAMP measured (Methods). Each point represents the mean ± range of cAMP values in duplicate wells of cells. Values are expressed as a percentage of the maximal cAMP attained. The TSH effective concentration required for half-maximal stimulation (EC50) is indicated by the horizontal dashed line. “V” represents a receptor with mutation to alanine of five charged residues (K244,E247,K250,E251 and R255). On this background, the alanine mutations of residue 244 or 247 were reverted to the wild-type. In the representative experiment (of four) shown, with basal cAMP levels of 0.2–0.4 pmoles per well of cells, mean maximum cAMP values were 19.2 (wild-type), 20.4 (K244A, E247A), 22.8 (“V”,A244K) and 19.5 (“V”,A247E) pmoles per well of cells.
Figure 7.
Schematic representation of the orientation of the TSHR LRD to the hinge region.
The crystal structure of the TSHR LRD in complex with the M22 human monoclonal stimulating autoantibody Fab [6](Protein Data Base 3G04) is shown as a ribbon diagram using FirstGlance in Jmol (http://molvis.sdsc.edu/fgij/). For mAb CS-17 to contact TSHR residues K244 and E247 on its convex surface, these residues cannot be buried in the hinge region and the TSHR LRD is, therefore, likely to tilt forward towards its concave face, as shown by the arrow. Obviously, the M22 IgG molecule including a second Fab and an Fc is even larger than the M22 Fab and would be even more susceptible to steric hindrance, with completion of its binding by forcing out water molecules leading to torsion of the LRD vis-a-vis the hinge region. It should be noted, however, that the M22 Fab alone can activate the TSHR [19]. For simplicity, TSHR amino acid residues K250, E251 and R255 interfacing with the hinge region are omitted and are better visualized in Figure 1. Of note, in Figure 1C (inferior view of the LRD base) these three residues lie towards one pole, with CS-17 epitope residues K244 and E247 at the opposite pole. This orientation supports the concept that the LRD is tilted forward in its interface with the hinge region.