Abstract
We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane 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 immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-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 covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.
Highlights
Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses
Based on the crystal structure of the GH (receptorj, complex [7] and associated physicochemical and biological evidence [8, 9], it is believed that the initial event in signal transduction by these receptors is hormone-induced oligomerization of receptor subunits, and this brings into proximity the proline-rich box 1 of the cytoplasmic domains, which is known to be essential for signal transduction [10,11,12]
We have recently found that herbimycin A, an inhibitor of JAK 2 kinase, is able to block a substantial portion of the prolactin signal to the promoter of the milk protein gene, J3-1actoglobulin, when expressed transiently with the prolactin receptor in CHO cells.f In order to define the role of tyrosine phosphorylation in prolactin stimulation of mammary gland function, we have examined hormone-stimulated tyrosine phosphorylation both in vivo, using mid-lactating rabbit mammary gland, and in vitro, using a stable CHO cell line expressing the full-length rabbit prolactin receptor
Summary
Vol 270, No 10, Issue of March 10, pp. 5136-5143, 1995 Printed in U.S.A. The Rabbit Mammary Gland Prolactin Receptor Is Tyrosinephosphorylated in Response to Prolactin in Vivo and in Vitro*. In the case of the prolactin receptor itself, some of these details are inferred, but there is biological evidence for receptor dimerization [22, 23], and rapid tyrosine phosphorylation of three proteins follows prolactin binding to the Nb2 lymphoma cell [24, 25] These three proteins comprise 97-kDa and 40-kDa components, which may be themselves kinases [25, 26], and a receptor-associated 121-kDa component, which appears to be JAK 2 [27, 28].
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