Waters M J, Daniel N, Bignon C, Djiane J
Unite d'Endocrinologie Moleculaire, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
J Biol Chem. 1995 Mar 10;270(10):5136-43. doi: 10.1074/jbc.270.10.5136.
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.
我们报告了首个体内研究，该研究证明了乳腺蛋白包括催乳素受体的酪氨酸磷酸化，这是对催乳素注射的反应。对乳腺膜提取物进行免疫印迹分析显示，在给予催乳素后5分钟内，200、130、115、100、90、70和45 kDa的亚基酪氨酸磷酸化增加。通过多种方法，包括免疫沉淀以及用针对催乳素受体的单克隆抗体（U5、917、110和82）和多克隆抗体（46）进行免疫印迹分析，确定100 kDa的成分是全长催乳素受体。在激素注射后1分钟内可见受体磷酸化达到最大值，并且为了获得强烈反应，有必要使兔子的内源性催乳素缺失36小时。通过在稳定转染兔催乳素受体cDNA的中国仓鼠卵巢细胞中进行验证，证实了全长受体的快速酪氨酸磷酸化。在体内和体外，磷酸化信号都是短暂的，在暴露于催乳素后10分钟内显著降低。通过用多克隆46对从转染的中国仓鼠卵巢细胞中免疫沉淀的受体进行免疫印迹分析，显示酪氨酸磷酸化的受体与JAK 2相关。在用[α-32P]叠氮基-ATP进行共价标记后，通过U5或多克隆46对受体复合物进行免疫沉淀，还显示一种48 kDa的ATP结合蛋白与乳腺受体相关。我们对催乳素受体酪氨酸磷酸化的证明增加了由受体/SH2蛋白相互作用调节信号通路的可能性，这将促进催乳素的特异性反应。需要一段激素缺失期才能实现显著的激素触发受体磷酸化这一事实表明，乳腺受体在体内主要处于脱敏状态，类似于相关的生长激素受体。
