THE RABBIT MAMMARY-GLAND PROLACTIN RECEPTOR IS TYROSINE-PHOSPHORYLATED IN RESPONSE TO PROLACTIN IN-VIVO AND IN-VITRO

We report the first in vivo study demonstrating tyrosine phosphorylati on of mammary gland proteins including the prolactin receptor, in resp onse to the injection of prolactin. Immunoblotting of mammary gland me mbrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunop recipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal rec eptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of the ir endogenous prolactin for 36 h, Rapid tyrosine phosphorylation of th e full-length receptor was verified by its demonstration in Chinese ha mster ovary cells stably transfected with rabbit prolactin receptor cD NA. Both in vivo and in vitro, the phosphorylation signal was transien t, being markedly reduced within 10 min of exposure to prolactin. Tyro sine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following c ovalent labeling with [alpha-P-32]azido-ATP. Our demonstration of prol actin receptor tyrosine phosphorylation raises the possibility of sign aling pathways regulated by receptor/SH2 protein interaction, which wo uld facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered recep tor phosphorylation indicates that the mammary gland receptor exists i n a largely desensitized state in vivo, analogous to the related growt h hormone receptor.