DESENSITIZATION OF ADENYLATE-CYCLASE IN CHINESE-HAMSTER OVARY CELLS TRANSFECTED WITH HUMAN THYROID-STIMULATING HORMONE-RECEPTOR

Desensitization or decreased response to the same (homologous) or othe r stimuli (heterologous) is a well known process. Homologous desensiti zation to TSH has been demonstrated in normal thyroid tissue. Chinese hamster ovary cells (CHO) transfected with normal human TSH receptor ( hTSHR) DNA, in contrast, have been reported not to desensitize. The pu rpose of our investigation was to determine whether CHO cells transfec ted with hTSHR desensitize in response to TSH and postreceptor stimula tion. CHO cells were stably transfected with plasmid DNA containing hT SHR; nontransfected CHO cells served as the control. TSH (10 mU/ml), 5 '-beta,gamma-imido-triphosphate [Gpp(NH)p; 0.1 mM], sodium fluoride (N aF; 10 mM), forskolin (10 mu M), and (Bu)(2)cAMP (100 mu M) were used to determine whether homologous or postreceptor heterologous desensiti zation of adenylate cyclase activity occurred in CHO-transfected cells . Intracellular cAMP accumulation was determined by RIA. Cells were in cubated with TSH (to stimulate TSH receptor), Gpp(NH)p, NaF (to stimul ate G-protein), forskolin (to stimulate adenylate cyclase activity), a nd (Bu)(2)cAMP (nonmetabolized cAMP analog). A second incubation was c arried out with TSH (10 mU/ml). Maximal desensitization to either TSH or postreceptor stimulation was observed at 2 h. When transfected CHO cells were preexposed to TSH (10 mU/ml) for 4 h, even the smallest dos e of TSH (0.001 mU/ml) caused desensitization. All substances that inc reased the intracellular cAMP concentration, such as TSH, Gpp(NH)p, Na F, forskolin, and (Bu)(2)cAMP, caused desensitization. The decrease in the cAMP response to TSH added in the second incubation was 63% less than the initial response to TSH or to postreceptor stimulation (P = 0 .0001). In conclusion, desensitization of hTSHR-transfected CHO cells occurs in response to both receptor and postreceptor stimulation that increase cAMP levels. Because hTSHR transfected CHO cells desensitize, no specific thyroid factor(s) other than increased levels of cAMP is required.