The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding.

Abstract

The hyt/hyt mouse is hypothyroid because of a mutation in the TSH receptor (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-coupled seven-transmembrane family of receptors. Insertion of this mutation into the wild-type rat receptor eliminated TSH binding and receptor function in transfected 293 and COS cells. Wild-type TSH-R conferred a 7.4-fold increase in cAMP and a 2.3-fold stimulation of a cAMP-responsive reporter gene. The P556L mutant receptor elicited no increase in cAMP or the reporter gene. Cells transfected with wild-type receptor bound TSH with a Kd 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 TSH, additional studies were performed to examine receptor processing and cellular localization. Mutant receptors from solubilized membranes also failed to bind TSH, indicating that the absence of binding to intact cells was not accounted for intracellular trapping of the mutant receptor. Western blot analyses demonstrated that the mutant and wild-type receptors were processed through a similar series of precursors and that a mature 95-kilodalton form of the mutant TSH-R was produced, consistent with its insertion into the plasma membrane. Immunofluorescence studies confirmed expression of the P556L mutant on the cell surface of transfected cells and in thyroid tissue from hyt/hyt mice. Although the extracellular domain of the TSH-R is sufficient for high affinity binding of TSH, we conclude that the hyt mutation in the fourth transmembrane domain eliminates TSH binding. These results suggest interactions between the extracellular and transmembrane domains of the TSH-R and indicate that this highly conserved proline is required for normal receptor structure and function.

Duox expression and related H2O2 measurement in mouse thyroid: onset in embryonic development and regulation by TSH in adult

“…Mice homozygous for the mutation have a normally located but hypoplastic and poorly functioning thyroid (Beamer et al 1981, Stein et al 1994, Gu et al 1995. In western blot experiments, Tshr hyt/hyt thyroids exhibited one wide immunoreactive band corresponding to protein(s) of approximately 150 kDa molecular mass ( Fig.…”

Differences in DBA/1J and DBA/2J reveal lipid QTL genes

“…Testing a hypomorphic strain for Tshr revealed that this gene does indeed have a strong effect on both TG and HDL levels. The mutant Tshr hyt strain has a Pro556Leu mutation (19,20) that makes it hypothyroid with decreased TG and increased HDL relative to controls. In our QTL, D2 alleles give rise to both increased TG and increased HDL, so the effect is not quite the same.…”

“…Animal protocols were reviewed and approved by the Animal Care and Use Committee at The Jackson Laboratory. Tshr hyt was originally identified as a spontaneous hypothyroid mutation in an RF/J mouse and was subsequently backcrossed into a BALB/cByJ background (N 10 ) and maintained through heterozygous 3 wild-type sib mating, because homozygotes are infertile (19,20). A strain deficient in villin1 (Vil1), a protein expressed in the brush border of the intestine, was generated at the Institut Curie (21) by villin1 gene targeting in 129S2/SvPas ES cells followed by injection into C57BL6 blastocysts; chimeric mice were subsequently intercrossed with DBA/2 mice to generate homozygous Vil1 2/2 mice on a mixed 129S2, B6, and D2 background.…”

Rescue from Dwarfism by Thyroid Function Compensation in rdw Rats

“…The hyt/hyt thyroid is a very small bi-lobed gland situated in the normal position [1,22]. The causal gene of hypothyroidism has been identified as Tg in cog/cog [11] and the TSH receptor [8] in hyt/hyt.…”