Tyroglobulin (Tg) depletion in receptor associated protein (RAP) KO mice is due to a reduction of Tg aggregates
Simonetta Lisi, Roberta Botta, Aldo Pinchera, Claudio Marcocci & Michele Marinò
RAP KO mice have a reduction of colloidal Tg resulting in subclinical hypothyroidism and histological signs of goiter. The difference in colloidal Tg between RAP KO and WT mice was striking by immunohistochemistry, but could not be detected in thyroid extracts. To explain this discrepancy, we hypothesized that the reduction of Tg reflected a reduction of Tg aggregates discarded during tissue extraction. To investigate this possibility, pellets obtained by thyroid homogenization were solubilized with 6M guanidine and analyzed by Western blotting. Tg resolved into two bands at 660 and 330 kDa, which were found in WT, but not in RAP KO mice, supporting a reduction of Tg aggregates in the latter. We then investigated the effects of detergents, denaturation and pH on homogenates separated into membrane-associated and cytoplasmic fractions. The Tg bands were detected in all samples from RAP KO and WT mice. Detergents and high pH increased the intensity of the bands in the cytoplasmic fractions from WT mice, suggesting the presence of Tg aggregates of high molecular mass. Under denaturing conditions the Tg bands were less intense, probably due to Tg degradation. In RAP KO mice, cytoplasmic Tg was less sensitive to detergents and pH, possibly because of a reduced number of Tg aggregates compared with WT mice. Higher amounts of Tg were found in the membrane-associated than in the cytoplasmic fractions, regardless of the extraction procedure and the genotype, representing Tg-containing vesicles within the colloid, Tg within intracellular organelles, and cell membrane-bound Tg. In RAP KO mice the amounts of membrane-associated Tg were greater than in WT mice, in agreement with immunohistochemical findings. In conclusion, the absence of RAP in the thyroid gland results in a reduction of colloidal Tg aggregates, which are known to represent the major storage form of thyroid hormones.