ACTIVATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES IN FRTL-5 THYROID-CELLS EXPRESSING A CONSTITUTIVELY ACTIVE GS-ALPHA

The expression of a constitutively activated Gs alpha protein in the r at thyroid cell line FRTL-5 causes an increase in the hormone-independ ent adenylyl cyclase activity and promotes TSH-independent growth of t he cells. In spite of the constitutive activation of the adenylyl cycl ase, the basal cAMP levels in these cells are only marginally increase d. To define the role of phosphodiesterases (PDEs) in the genesis of t his phenotype, cyclic nucleotide hydrolysis was determined in two cell lines expressing a mutated Gs alpha (Q227L). In these cells, the hydr olysis of both cAMP and cGMP was markedly increased in comparison with normal cells. This increase is the result of the activation of differ ent forms of PDEs. Analysis of the cGMP hydrolysis and Ca++/calmodulin stimulation of the PDE activity indicated that the activity of a Ca+/calmodulin stimulated PDE is increased in both cell lines. In additio n, an increase in high-affinity, rolipram-sensitive cAMP-PDE activity was associated in both cell lines with the appearance of a 67-68 kilod alton (kDa) protein that cross-reacts with two antibodies against cAMP -PDEs. This form had the properties of ratPDE3.2/PDE4D2, a cAMP-PDE th at is inducible by TSH in wild type cells. That an increase in cAMP-sp ecific, rolipram-sensitive PDE plays a role in the phenotype induced b y Q227L Gs alpha was confirmed by measurements of the mitogenic activi ty. Incubation with rolipram, which had no effect on wild type cells, caused an increase in cAMP levels and further stimulated TSH-independe nt proliferation in both cell lines carrying the mutation. These data demonstrate that activation of the PDE system in FRTL-5 counteracts, a t least in part, the phenotype caused by an activating mutation in Gs alpha.