ABSTRACT
Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor alpha (IL-3Ralpha), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with beta(IL-3) mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPalpha and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.
Subject(s)
Alternative Splicing , Cell Differentiation , Interleukin-3 Receptor alpha Subunit/metabolism , Receptors, Interleukin-3/metabolism , Signal Transduction , Animals , Blotting, Western , COS Cells , Cell Proliferation , Cells, Cultured , Chlorocebus aethiops , Flow Cytometry , Glycosylation , Humans , Interleukin-3 Receptor alpha Subunit/genetics , Janus Kinase 2/metabolism , Mice , Mice, Transgenic , MicroRNAs/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Isoforms , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Interleukin-3/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/metabolism , Transcription Factors/metabolismABSTRACT
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).
Subject(s)
Cytokine Receptor Common beta Subunit/physiology , Polysaccharides/physiology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Receptors, Interleukin-3/physiology , Receptors, Interleukin-5/physiology , Animals , COS Cells , Chlorocebus aethiops , Cytokine Receptor Common beta Subunit/chemistry , Cytokine Receptor Common beta Subunit/genetics , Humans , Interleukin-3/metabolism , Interleukin-5/metabolism , Mice , Mutagenesis, Site-Directed , Polysaccharides/chemistry , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Receptors, Interleukin-3/chemistry , Receptors, Interleukin-5/chemistryABSTRACT
Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human beta-common (hbetac) receptor and the murine IL-3-specific (beta(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr(15) and Phe(79), of the hbetac receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of hbetac, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the beta(IL-3) receptor is also a homodimer. Its high sequence homology with hbetac suggests that their structures are homologous, and we identified an analogous binding interface in beta(IL-3) for direct IL-3 binding to the high affinity binding site in hbetac. Tyr(21) (A-B loop), Phe(85), and Asn(87) (E-F loop) of domain 1; Ile(320) of the interdomain loop; and Tyr(348) (B'-C' loop) and Tyr(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg(84) and Tyr(317) major secondary roles in direct murine IL-3 binding to the beta(IL-3)receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 alpha receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hbetac.
Subject(s)
Interleukin-3/metabolism , Receptors, Cell Surface/chemistry , Alanine/chemistry , Amino Acid Sequence , Animals , COS Cells , Cell Division , Cross-Linking Reagents/pharmacology , Crystallography, X-Ray , Cytokine Receptor Common beta Subunit , Cytokines/metabolism , DNA, Complementary/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Epitopes , Flow Cytometry , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-5/metabolism , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/metabolism , Sequence Homology, Amino Acid , Transfection , UltracentrifugationABSTRACT
The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific alpha-subunits and a common beta-subunit (betac), the major signaling entity. The way in which betac interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of betac revealed a possible ligand-binding interface composed of domain 1 of one chain of the betac dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr(15) (A-B loop) and Phe(79) (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-5/pharmacology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Receptors, Interleukin-3/chemistry , Receptors, Interleukin/chemistry , Epitopes , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-5/metabolism , Protein Subunits , Receptors, Interleukin-5 , Structure-Activity RelationshipABSTRACT
UNLABELLED: Sympathetic hyperactivity during sudden intracranial hypertension leads to cardiovascular instability, myocardial dysfunction, and neurogenic pulmonary edema. Because spinal anesthesia is associated with sympatholysis, we investigated the protective effects of intrathecal lidocaine in a rodent model. Halothane-anesthetized rats were given a 10-microL intrathecal injection of saline (n = 10) or lidocaine 1% (n = 6). A subdural balloon catheter was inflated for 60 s to produce intracranial hypertension. Hemodynamics were monitored, and hearts and lungs were harvested for histological examination. In Saline versus Lidocaine-Treated rats, peak mean arterial blood pressure during balloon inflation was 115 +/- 4 mm Hg versus 78 +/- 8 mm Hg (P < 0.05), mean arterial blood pressure 30 min after balloon deflation was 47 +/- 2 mm Hg versus 67 +/- 3 mm Hg (P < 0.05), and lung weight was 1.54 +/- 0.03 g versus 1.41 +/- 0.04 g (P < 0.05), respectively. Cardiac dysrhythmias and electrocardiographic changes were more frequent in the Saline-Treated group (P < 0.05). Saline-Treated rats had extensive, hemorrhagic pulmonary edema, whereas the Lidocaine-Treated rats had only patchy areas of lung abnormality. Histological changes in the myocardium were rare, and no difference was found between the two groups. We conclude that intrathecal lidocaine prevents cardiovascular collapse and neurogenic pulmonary edema in a rat model of acute intracranial hypertension. IMPLICATIONS: In a rat model of intracranial balloon inflation, intrathecal lidocaine prevented cardiovascular collapse and neurogenic pulmonary edema. Descending neural pathways are involved in the development of cardiopulmonary complications associated with acute intracranial hypertension.