Prolactin receptor and signal transduction to milk protein genes.
After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (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 single species of 100 kDa. Using these cells, we analyzed the effects of PRL 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. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 microM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the beta lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.
Unité d'Endocrinologie Moléculaire, INRA Jouy en Josas, France.
J
J
Djiane
N
N
Daniel
C
C
Bignon
J
J
Paly
M
M
Waters
P
P
Vacher
B
B
Dufy
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008894:Milk Proteins / Q000235:genetics*; D011388:Prolactin / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D018076:DNA, Complementary; D008894:Milk Proteins; D011981:Receptors, Prolactin; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; D011505:Protein-Tyrosine Kinases; D002118:Calcium