Identification and functional characterization of two novel activating thyrotropin receptor mutants in toxic thyroid...

Two previously unreported thyrotropin (TSH) receptor mutations, A623F and I635V, were identified in toxic follicular thyroid adenoma specimens from two patients with hyperthyroidism. Our aim was to characterize both novel mutants in terms of the following: cAMP basal constitutive activity, cAMP response to TSH, plasma membrane expression levels, and TSH binding properties.

We performed DNA extraction for TSHR gene sequencing. COS-7 cells were transiently transfected with wild-type and mutated TSH receptor constructs for determination of basal cAMP constitutive activity and dose-response accumulation of cAMP using recombinant human TSH. Flow cytometry analysis was performed to evaluate plasma membrane expression. Binding studies using bovine TSH as a ligand were performed to compare the affinities of wild-type and mutated TSH receptors for TSH.

Both mutants, A623F and I635V, had higher cAMP basal constitutive activities than the wild-type TSH receptor. A623F but not I635V showed lower plasma membrane expression than the wild-type receptor. IC50, an indirect measurement of ligand-receptor affinity, was lower in A623F and higher in I635V than in the wild-type TSH receptor, although no statistically significant differences were observed. No differences were observed in EC50 and although the absolute values of maximal stimulation achieved with both mutants were higher than the wild type, the differences did not achieve statistical significance.

A623F and I635V are two naturally occurring TSH receptor mutations that increase basal cAMP accumulation and consequently promote the development of toxic follicular thyroid adenoma. cAMP response to increasing TSH dose is retained by A623F and I635V mutated receptors and the maximal stimulation obtained is not different from that of the wild-type receptor. Substitution of alanine 623 by phenylalanine 623 at the third intracellular loop of the TSH receptor decreases its plasma membrane expression, indicating that alanine 623 is important in directing the TSH receptor to the cell surface or in down-regulating the constitutive receptor. By contrast, isoleucine 635, located in the sixth transmembrane domain, is important in regulating TSH receptor basal activity but does not modify its plasma membrane expression.