The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNgamma-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.
An eight-amino-acid synthetic peptide (IIe1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C with a ratio of 60:40. At 30 degrees C, the two peaks coalesced into a single peak The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its ID NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25-70 degrees C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK 506-binding protein (FKBP) serves as an on-off switch for cytokine receptor activation.
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