Objective: Constitutively activating mutations of the thyrotropin receptor (TSHR) have been found in the majority of autonomously functioning thyroid nodules (AFTNs) in European patients. The reported frequency of these mutations varies among reports but amounts to 50 -80%. To date, only one such mutation responsible for AFTNs has been identified in the Japanese population and the pathogenic role of such mutations in Japanese AFTNs has been questioned. In the present study, we evaluated the frequency of activating mutations in the TSHR and Gas in 10 Japanese AFTNs. Design: Genomic DNA was extracted from fresh frozen tissue. The TSHR and the almost entire sequence of the gene coding for the a subunit of Gs have been amplified and sequenced. Results: In sequence analysis, four mutations in the TSHR (T632A, I486M, M453T and L512R) were found. To complete our analysis, we searched mutations in the gene coding for the a subunit of Gs, in the samples negative for TSHR mutations. In one case a mutation (R201H) affecting GTPase activity was found. Conclusions: If we focus on the solitary nodules, we obtain the same mutation proportion as in European patients (70%). The absence of TSHR and Gas mutations in a significant proportion of autonomous adenomas in multinodular goiters suggests that other causes may also play a role in the genesis of these lesions.

Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the alpha- subunit of the heterotrimeric G(s) and G(q) proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mm methyl-beta-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.

Objective: The cyclic AMP (cAMP) cascade is the main regulatory pathway in thyrocytes. Whilst activating mutations in the TSH receptor or in the Gs a-subunit, which increase cAMP levels, have been shown to be responsible for 80% of the autonomous adenomas, no such mutations have been observed in the other types of thyroid tumors, suggesting that other mechanisms exist. The discovery of Epac (`exchange nucleotide protein directly activated by cAMP'), a novel cAMP-binding protein, which is strongly expressed in the thyroid, raised the possibility of a role for this protein in the generation of the unexplained cold thyroid follicular adenomas. Thus, we investigated whether activating mutations in either Epac or Rap (the downstream target of Epac) could be responsible for the generation of these thyroid nodules. Design: Epac and Rap1 (Rap1A and Rap1B) cDNAs were sequenced in 10 patients. The sequencing of the cDNAs was realized on both strands in the cold nodule and the juxtanodular tissue of each patient. Results: No mutations in either Epac or Rap1 cDNAs were found. For five patients, a polymorphism in Epac at codon 332 (Gly±Ser) was observed. Conclusions: In this report, we show that the cAMP±Epac±Rap1 signaling pathway in the thyroid gland does not play a major role in the generation of cold thyroid follicular adenomas, since no mutations in either Epac or Rap1 could be observed in the 10 nodules studied.

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