Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

Prolactin (PRL) has been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2. The present study represents an initial effort to identify the phosphorylation repertoire of the PRL receptor (PRLR). For this purpose we have modified the rat PRLR cDNA to encode an additional N-terminal epitope specifically designed to allow the rapid purification of the PRLR and associated proteins from transfected cells. The Flag-tagged PRLR was stably expressed in the human 293 cell line. PRL induced tyrosine phosphorylation of proteins of 85, 95, and 185 kDa from 10 to 30 min after PRL stimulation. Immunoblot analysis of immunoprecipitation indicates that p85 corresponds to the 85-kDa regulatory subunit of phosphatidylinositol (PI)-3 kinase, p95 to PRLR, and p185 to insulin receptor substrate 1 (IRS-1). Both PI-3 kinase and IRS-1 appear to associate with PRLR in a PRL-dependent manner. These results thus indicate that kinases other than JAK2, namely PI-3 kinase, are activated by PRL. PRL1 binding to its cell surface receptor initiates a series of molecular interactions that ultimately determines the specific physiological response. Following PRL stimulation, PRL receptors are tyrosyl-phosphorylated; recent efforts to identify signal transducers activated by the PRL receptor have demonstrated that PRLR associates with and activates two cytoplasmic tyrosine kinases of the Janus T-K family (1-4), although JAK2 appears to be the major kinase involved in most responses.A number of signaling molecules form stable complexes with other tyrosyl-phosphorylated receptors via an SH2 domain, including insulin receptor. The rationale for sharing common intracellular pathways between IR and PRLR is further substantiated by the insulin-like effect of growth hormone and to a lesser extent of PRL in a variety of cell types (5-7). These include 1) increased glucose-stimulated insulin secretion and decreased threshold of glucose stimulation (8), 2) increased insulin synthesis (9), 3) increased ␤-cell proliferation (10), and, more recently, it has been proposed that lactogenic hormones are primarily responsible for the enhanced islet function observed during pregnancy (11). To facilitate detection of the interaction repertoire and the phosphorylation repertoire of the PRLR, we have modified the rat PRLR cDNA to encode an additional N-terminal epitope specifically designed to allow the rapid purification of the PRLR and associated proteins from transfected cells. Our results clearly show that PRLR associates with insulin receptor substrate 1 and PI-3Ј kinase. Upon PRL stimulation, both association with PRLR and tyrosyl phosphorylation of these two proteins are activated. EXPERIMENTAL PROCEDURESReagents and Antibodies-Ovine PRL was a gift from the National Hormone and Pituitary/NIDDK program (Baltimore, MD). The antiphosphotyrosine antibody (␣PY), monoclonal IgG 2 bk antiserum to the 85-kDa subunit of PI-3Ј kinase, and rabbit polyclonal antibody to IRS-1 (anti-rat C-terminal) were purchased from Upstat...

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Cytokine receptor signaling involves the Jak/Stat pathways. Heterotrimeric IL-2R (K K, L L, Q Q chains) activates Jak1 and Jak3, whereas homodimeric PRLR activates Jak2. The requirements directing such specificity of Jak activation are unknown. We show that chimeric receptors containing the intracellular domain of IL-2RL L chain fused to the extracellular domain of either EPOR or Kit, a non-cytokine receptor, activate Jak2. This observation provides evidence that IL-2RL L intrinsically possesses the ability to activate Jak2, but that this property is only displayed in homodimerized complexes. Our data suggest a role for the stoichiometry of cytokine receptors in selective activation of Janus kinases.z 1998 Federation of European Biochemical Societies.