Rab proteins are small GTPases that control distinct vesicular transport steps. Along the endocytic pathway, Rab5a is a ratelimiting catalyst of internalization, and Rab7 controls trafficking through late endosomes to lysosomes. The dependence of thyroid hormone production by thyrocytes on thyroglobulin endocytosis and intracellular processing in late endosomes͞lysosomes suggests that its rate can be regulated by the expression or function of these endocytic catalysts. We compared the expression level and membrane recruitment of Rab5a and Rab7 in autonomous thyroid adenomas (where the cAMP cascade is constitutively activated) and surrounding quiescent tissues. The concentrations of Rab5a and Rab7, but not of Rab8, were coordinately increased up to 6-fold in adenomas, and correlated with a proportionate decrease in soluble thyroglobulin content (reflecting colloid depletion by accelerated endocytic uptake in hyperactive tissue). In adenomas, a higher proportion of Rab5a and Rab7 was membrane associated, and the equilibrium density of particulate Rab7 and iodine shifted toward lysosomal fractions, indicating that progression along the degradation pathway also was promoted. In cultures of polarized human thyrocytes from normal patients, thyroid-stimulating hormone or forskolin increased, to a similar extent, Rab5a and Rab7 but not Rab8 expression, apical endocytosis of thyroglobulin and lucifer yellow, and basolateral secretion of T 3 and T4. Taken together, these in vivo and in vitro observations demonstrate that thyroid-stimulating hormone, via cAMP, coordinately enhances the expression of Rab5a and Rab7, which promote Tg endocytosis and transfer to lysosomes, respectively, resulting in accelerated thyroid hormone production.

Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid hormone secretion depends on apical endocytosis of thyroglobulin (Tg), we investigated whether ClC-5 is expressed in the thyroid and affects its function, using Clcn5-deficient knockout (KO) mice. We found that ClC-5 is highly expressed in wild-type mouse thyroid ( approximately 40% of mRNA kidney level). The protein was immunolocalized at the apical pole of thyrocytes. In Percoll gradients, ClC-5 overlapped with plasma membrane and early endosome markers, but best codistributed with the late endosomal marker, Rab7. ClC-5 KO mice were euthyroid (normal T4 and TSH serum levels) but developed a goiter with parallel iodine and Tg accumulation (i.e. normal Tg iodination level). When comparing ClC-5 KO with wild-type mice, thyroid 125I uptake after 1 h was doubled, incorporation into Tg was decreased by approximately 2-fold, so that trichloroacetic acid-soluble 125I increased approximately 4-fold. Enhanced 125I- efflux upon perchlorate and presence of 125I-Tg as autoradiographic rings at follicle periphery demonstrated delayed iodide organification. Endocytic trafficking of 125I-Tg toward lysosomes was not inhibited. Expression of pendrin, an I-/Cl- exchanger involved in apical iodide efflux, was selectively decreased by 60% in KO mice at mRNA and protein levels. Thus, ClC-5 is well expressed in the thyroid but is not critical for apical endocytosis, contrary to the kidney. Instead, the goiter associated with ClC-5 KO results from impaired rate of apical iodide efflux by down-regulation of pendrin expression.

This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.