SPECIFIC DNA-BINDING OF STAT5, BUT NOT OF GLUCOCORTICOID RECEPTOR, ISREQUIRED FOR THEIR FUNCTIONAL COOPERATION IN THE REGULATION OF GENE-TRANSCRIPTION

Prolactin and glucocorticoid hormone are signals which regulate the tr anscription of milk protein genes in mammary epithelial cells. We have investigated the molecular mechanisms by which these hormones coopera te in the induction of transcription, Both hormones activate latent tr anscription factors in the cytoplasm of mammary epithelial cells, Prol actin exerts its effect through binding to the extracellular domain of the prolactin receptor and through receptor dimerization, This leads to the activation of a protein tyrosine kinase (Jak2), which is noncov alently associated with the cytoplasmic domain of the prolactin recept or, Jak2 phosphorylates the signal transducer and transcription activa tor (Stat5) which causes its dimerization and nuclear translocation wh ere Stat5 specifically binds to sequence elements in the promoter regi ons of milk protein genes. In comparison, the glucocorticoid receptor is activated by a lipophilic steroid ligand in the cytoplasm which cau ses allosteric changes in the molecule, dimerization, and nuclear loca lization. It has been demonstrated that Stat5 and the glucocorticoid r eceptor form a molecular complex which cooperates in the induction of transcription of the beta-casein gene. We have defined the DNA sequenc e requirements for this cooperative mechanism and have delimited the f unctional domains in Stat5 and the glucocorticoid receptor that are ne cessary for the functional interaction, We find that the Stat5 respons e element (Stat5RE) within the beta-casein gene promoter is sufficient to elicit the cooperative action of Stat5 and the glucocorticoid rece ptor on transcription. Activation of Stat5 through phosphorylation of tyrosine 694 is an absolute prerequisite for transcription, Deletion o f the transactivation domain of Stat5 results in a molecule which cann ot mediate transactivation by-itself but can still cooperate with the glucocorticoid receptor, Mutated variants of the glucocorticoid recept or with a nonfunctional DNA binding domain or a DNA binding domain con tributed by the estrogen receptor are still able to cooperate with Sta t5 in transcriptional induction, Deletion of the ligand binding domain of the glucocorticoid receptor does not impede cooperation with Stat5 , whereas deletion of the AF-1 transactivation domain does prevent coo peration, Our results indicate that the glucocorticoid receptor acts a s a ligand-dependent coactivator of Stat5 independently of its DNA bin ding function.