Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function.
A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.
D000262:Adenylyl Cyclases / Q000235:genetics; D000818:Animals; D002116:Calcitonin / Q000494:pharmacology*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004351:Drug Resistance; D006822:Hybrid Cells; D009154:Mutation; D010960:Plasminogen Activators / Q000096:biosynthesis*; D011956:Receptors, Cell Surface / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis; D014667:Vasopressins / Q000494:pharmacology*
D011956:Receptors, Cell Surface; D014667:Vasopressins; D002116:Calcitonin; D000242:Cyclic AMP; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D000262:Adenylyl Cyclases