Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity

Constitutively activating mutations in the human thyroid-stimulating hormon e (TSH) receptor (TSHr) have been identified as the most prevalent molecula r cause of non-autoimmune hyperthyroidism. To investigate the feasibility o f an animal model for non-autoimmune hyperthyroidism, we introduced two mut ations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, bas al cAMP and IP accumulation, in the human TSHr, the lie 486 Phe mutation ca uses a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signalin g. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2. 4 kb PCR product from the mouse TSHr was cloned into the pGEM-T Vector syst em. lie was substituted with Phe at codon 486 and Val with Ala at codon 509 , These mutated mouse TSHrs were subcloned in the pSVL expression vector. A fter transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined a nd directly compared to the human TSHr, Whereas constitutively activating m utations of the human parathyroid hormone (PTH)/PTH-related peptide recepto r showed little or no change in basal cAMP accumulation when introduced int o the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in co nstitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimm une hyperthyroidism by homologous recombination to study the pathogenetic m echanisms of non-autoimmune hyperthyroidism.