GENE USAGE AND REGULATION OF GSA GENE-EXPRESSION IN THYROID-CELLS

The TSH receptor is a C-protein-coupled seven transmembrane segment re ceptor. The interaction between TSH and its receptor mediates signal t ransduction by activating adenylyl cyclase through Gs alpha. There are four forms of Gs alpha (two short [45 kDa] and two large [52 kDa]), a rising from alternative splicing of exon 3 of the Gs alpha gene. Gs al pha-1 and -2 contain exon 3, whereas exon 3 is spliced out in Gs alpha -3 and -4. The inclusion of a serine residue at the 3' splice junction of exon 3 distinguishes Gs alpha-2 and -4 from Gs alpha-1 and -3, The expression of different Gs alpha forms appears to be tissue-specific. In this study, we have examined the Gs alpha splice variants in 26 hu man thyroid tumor specimens and rat thyroid tissues as well as a rat F RTL-5 cell line. Furthermore, we have studied the regulation of the Gs alpha gene expression by TSH and cAMP in FRTL-5 cells. We found that Gs alpha-1 and -4 mRNA were present in both human and rat thyroid cell s, although Gs alpha-4 was more abundant in human thyroid cells as com pared to rat thyroid and FRTL-5 cells. The Gs alpha mRNA can be easily amplified by RT-PCR regardless of tumor type and stage, suggesting th at Gs alpha gene expression in thyroid tumors may not be markedly affe cted by dedifferentiation of thyroid cells. Both TSH and 8-bromo-cAMP, a cAMP analog, can stimulate the Gs alpha gene expression in FRTL-5 c ells with maximal effect by 6 h and 1 h, respectively. The addition of cycloheximide to the culture of FRTL-5 cells abolished the effect of bTSH, but not that of 8-bromoc-AMP, on the expression of the gs alpha gene. Cellular cAMP measurements showed that bTSH-stimulated cAMP prod uction was significantly reduced to the basal level after addition of cycloheximide, These results suggest that regulation of the Gs alpha g ene expression by TSH is mediated by a cAMP-dependent process and requ ires new protein synthesis.