Alteration of the functional activity of G s protein in thyrotropin-desensitized pig thyroid cells

Abstract

Changes in the sensitivity of adenylyl cyclase observed in pig thyroid cells cultured 2 days in the presence of thyroid-stimulating hormone (TSH) or forskolin were assessed by examining the properties of G s protein. Chronic treatment of thyroid cells with various concentrations of TSH (0.01–1 mU/ml) or forskolin (0.1–10 μM) increased the response of adenylyl cyclase to a further stimulation by forskolin or NaF + AlCl 3 ([A1F 4] −). In contrast, the enzyme activation promoted by guanosine 5′-(β,γ-imido) triphosphate (Gpp(NH)p) was markedly affected. There was a significant increase in adenylyl cylcase activation by Gpp(NH)p in membranes from cells treated with low concentrations of TSH (≤ 0.1 mU/ml) or forskolin (≤ 1 μM) but a significant decrease in membranes from cells cultured with a higher concentration of TSH (1 mU/ml) or forskolin (10 μM). This decrease in Gpp(NH)p-stimulated adenylyl cyclase activity was mimicked by 8-bromo-cAMP but not by 1,9-dideoxyforskolin, a forskolin analogue which has lost its ability to activate adenylyl cyclase. There was a good correlation with the ability of G s protein to be ADP-ribosylated by cholera toxin: labeling of G s protein decreased following chronic treatment of thyroid cells with TSH (1 mU/ml) or forskolin (10 μM). In contrast, under the same experimental culture conditions a slight but significant increase in the quantity of G s subunits was observed by immunoblotting analysis. These results indicate: (i) that the reduced functional activity of G s induced by the culture of cells with TSH is mediated by a cAMP-dependent mechanism, (ii) that this alteration is not correlated to a defect in the synthesis of G s protein.