PROLACTIN RECEPTOR AND SIGNAL-TRANSDUCTION TO MILK PROTEIN GENES

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a fu nctional assay was established using co-transfection of PRL receptor c DNA together with a milk protein promoter/chloramphenicol acetyl trans ferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a singl e species of 100 kDa. Using these cells, we analyzed the effects of PR L on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. W e have also analyzed tyrosine phosphorylation induced by PRL. In CHO c ells stably transfected with PRL receptor cDNA, PRL induced a very rap id and transient tyrosine phosphorylation of a 100-kDa protein which i s most probably the PRL receptor. The same finding was obtained in mam mary membranes after PRL injection to lactating rabbits. Whereas tyros ine kinase inhibitors genistein and lavendustin were without effect, P RL stimulation of milk protein gene promoters was partially inhibited by 2 mu M herbimycin in CHO cells co-transfected with PRL receptor cDN A and the p lactoglobulin CAT construct. Taken together these observat ions indicate that the cytoplasmic domain of the PRL receptor interact s with one or several tyrosine kinases, which may represent early post receptor events necessary for PRL signal transduction to milk protein genes.