Prolactin (PRL) has been shown to activate the cytoplasmic tyrosine kinase Janus kinase 2 (Jak2) and the subsequent recruitment of various signaling molecules including members of the signal transducer and activator of transcription family of transcription factors. Recently, an expanding family of cytokine-inducible inhibitors of signaling has been identified that initially included four members: suppressor of cytokine signaling (SOCS)-1, SOCS-2, SOCS-3, and cytokine-inducible src homology domain 2 (SH-2) proteins. The present study analyzes the role of these members in PRL signaling. Constitutive expression of SOCS-1 and SOCS-3 suppressed PRL-induced signal transducer and activator of transcription 5-dependent gene transcription, and Jak2 tyrosine kinase activity was greatly reduced in the presence of SOCS-1 or SOCS-3. SOCS-1 was shown to associate with Jak2, whereas SOCS-2 was associated with the prolactin receptor. Co-transfection studies were conducted to further analyze the interactions of SOCS proteins. SOCS-2 was shown to suppress the inhibitory effect of SOCS-1 by restoring Jak2 kinase activity but did not affect the inhibitory effect of SOCS-3 on PRL signaling. Northern blot analysis revealed that SOCS-3 and SOCS-1 genes were transiently expressed in response to PRL, both in vivo and in vitro, whereas the expression of SOCS-2 and CIS genes was still elevated 24 h after hormonal stimulation. We thus propose that the early expressed SOCS genes (SOCS-1 and SOCS-3) switch off PRL signaling and that the later expressed SOCS-2 gene can restore the sensitivity of cells to PRL, partly by suppressing the SOCS-1 inhibitory effect.Prolactin (PRL) 1 exerts its effects via the PRL receptor (PRLR) and the activation of intracellular signaling molecules through the Jak-STAT pathway (1). Ligand binding leads to dimerization of the receptor and activation of Jak2, which in turn phosphorylates the PRL receptor and the transcription factor STAT5. However, less is known regarding how PRL signal transduction is switched off. In addition to Jak2 and STAT5 induction, PRL receptor activation also results in the stimulation of the protein tyrosine phosphatase SHP-2; however, in the case of the PRL receptor, a positive role in signal transduction has been assigned (2). Recently, a novel family of proteins capable of suppressing cytokine signal transduction has been identified that function in a classic negative feedback loop to regulate cytokine signaling (3-11). Expression of suppressor of cytokine signaling (SOCS)-1 suppresses interleukin 6-induced macrophage differentiation of murine myeloid leukemia cell line M1 and interleukin 6-induced receptor phosphorylation as well as STAT activation. SOCS-1 interacts with the catalytic region of Jak kinases and suppresses their tyrosine kinase activity and, as a result, the activation of STATs. Studies of mutated or deleted SOCS-1 proteins reported that the N-terminal domain and the SH-2 domain were required to inhibit leukemia-inhibitory factor signal transduction (12).SOCS-1 and SOCS...

Prolactin (PRL) interacts with a single chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers activation of Jak2 kinase which phosphorylates the PRL receptor itself and the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Selection of the particular substrate (Stat 5), that is characterized by transcriptional responses to PRL, has been shown to be determined by specific tyrosine-based motifs common to many cytokine receptors. PRL-induced activation of Stat5 was abolished in 293 fibroblasts expressing PRL receptor mutants lacking all intracellular tyrosines. We have identified tyrosine phosphorylation sites of the PRL receptor (residues 580, 479, and 473) necessary for maximal Stat5 activation and subsequent Stat5-dependent gene transcription. Moreover, we have shown that none of the tyrosine residues of the PRL receptor are implicated in activation of Jak2. This study demonstrates that only specific tyrosines in the PRL receptor are phosphorylated and are in fact utilized differentially for Stat5-mediated transcriptional signaling.

In addition to a long form of 591 amino acids (aa), two other forms of PRL receptor (PRLR), differing in the length of their cytoplasmic domains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal similar to the long form, whereas the short form of 291 aa is inactive. The ability of PRL to activate the promoter of the beta-casein gene or the lactogenic hormone responsive element fused to the luciferase reporter was assessed in Chinese hamster ovary cells or 293 fibroblasts transiently transfected with PRLR cDNAs. The function of the short form was examined after cotransfection of both the long and short forms. These results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2) activation. The present study also investigates the possible participation of cytoplasmic receptors in the signal transduction pathway, using cotransfection experiments and a new approach that selectively determines the contribution of cytoplasmic receptors in the process of signal transduction. We cotransfected Chinese hamster ovary cells with two cDNA constructs: a cytoplasmic (soluble) form of the receptor with a deleted signal peptide (delta-19), which is unable to bind PRL, and a functionally inactive receptor mutant (lacking box 1), which is anchored in the plasma membrane and able to bind PRL. This approach has allowed us to show that delta-19, lacking expression at the plasma membrane, can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing that association/activation occurs with a PRL-PRLR complex initiated at the cell surface level; box 1 of the cytoplasmic form is necessary to rescue this partial transcriptional activity of the inactive mutant. This partial recovery is also parallel to the partial activation of JAK2, indicating that the signal transduction pathway implicated JAK2. Our results provide evidence that heterodimerization of receptors can be implicated either in the positive or in negative activation of gene transcription.