I. CELL PROLIFERATION IN NORMAL THYROID TISSUE cles and of their supporting mesenchymal tissue and IN VIVO cells, the endothelial cells of the capillaries (20%) and the fibroblasts (10%) (78). Scarce calcitonin-secreting A. Physiological Situation parafollicular cells are located at the periphery of the follicles. After its differentiation in the fetus, the tissue The thyroid tissue is mainly composed of thyroid grows roughly in parallel with body weight and remains follicular cells, the thyrocytes (70954, arranged in folli-at the same size throughout adult life.

An approach based on the polymerase chain reaction has been devised to clone new members of the family of genes encoding guanosine triphosphate-binding protein (G protein)-coupled receptors. Degenerate primers corresponding to consensus sequences of the third and sixth transmembrane segments of available receptors were used to selectively amplify and clone members of this gene family from thyroid complementary DNA. Clones encoding three known receptors and four new putative receptors were obtained. Sequence comparisons established that the new genes belong to the G protein-coupled receptor family. Close structural similarity was observed between one of the putative receptors and the 5HT1a receptor. Two other molecules displayed common sequence characteristics, suggesting that they are members of a new subfamily of receptors with a very short nonglycosylated (extracellular) amino-terminal extension.

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Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAP-KII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N 6 -benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10 -100-fold. Phenylthio substituents in position 8, particularly with MeO-or Cl-in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT-and 2-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.