THE THYROTROPIN (TSH) RECEPTOR TRANSMEMBRANE DOMAIN MUTATION (PRO556-LEU) IN THE HYPOTHYROID HYT HYT MOUSE RESULTS IN PLASMA-MEMBRANE TARGETING BUT DEFECTIVE TSH BINDING/

The hyt/hyt mouse is hypothyroid because of a mutation in the TSH rece ptor (TSH-R). In this report, we confirm the presence of a Pro to Leu mutation in amino acid 556 of the fourth transmembrane domain (TM4) of the TSH-R. This Pro is highly conserved in members of the G protein-c oupled seven-transmembrane family of receptors. insertion of this muta tion into the wild-type rat receptor eliminated TSH binding and recept or function in transfected 293 and COS cells. Wild-type TSH-R conferre d a 7.4-fold increase in cAMP and a 2.3-fold stimulation of a cAMP-res ponsive reporter gene. The P556L mutant receptor elicited no increase in cAMP or the reporter gene. Cells transfected with wild-type recepto r bound TSH with a K-d of 3.3 x 10(-10) M, whereas no TSH binding was detected with the P556L mutant. Because the P556L mutation occurs in a receptor region (TM4) that is not expected to alter the binding of TS H, additional studies were performed to examine receptor processing an d cellular localization. Mutant receptors from solubilized membranes a lso failed to bind TSH, indicating that the absence of binding Do inta ct cells was not accounted for intracellular trapping of the mutant re ceptor. Western blot analyses demonstrated that the mutant and wild-ty pe receptors were processed through a similar series of precursors and that a mature 95-kilodalton form of the mutant TSH-R was produced, co nsistent with its insertion into the plasma membrane. Immunonuofluores cence studies confirmed expression of the P556L mutant on the cell sur face of transfected cells and in thyroid tissue from hyt/hyt mice. Alt hough the extracellular domain of the TSH-R is sufficient for high aff inity binding of TSH, we conclude that the hyt mutation in the fourth transmembrane domain eliminates TSH binding. These results suggest int eractions between the extracellular and transmembrane domains of the T SH-R and indicate that this highly conserved proline is required for n ormal receptor structure and function.