Multiple regions within the cytoplasmic domains of the leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells.

Abstract

The receptor for leukemia inhibitory factor (LIFR), in combination with the signal-transducing subunit for interleukin-6-type cytokine receptors, gp130, and LIF, activates transcription of acute-phase plasma protein genes in human and rat hepatoma cells and the vasoactive intestinal peptide gene in a human neuroblastoma cell line. To identify the regions within the cytoplasmic domain of LIFR that initiate signal transduction independently of gpl30, we constructed a chimeric receptor by linking the extracellular domain of the granulocyte colony-stimulating factor receptor (G-CSFR) to the transmembrane and cytoplasmic domain of human LIFR. The function of the chimeric receptor protein in transcriptional activation was assessed by G-CSF-mediated stimulation of cotransfected cytokine-responsive reporter gene constructs in hepatoma and neuroblastoma cells. By using the full-length cytoplasmic domain and mutants with progressive carboxyterminal deletions, internal deletions, or point mutations, we identified the first 150 amino acid residues of LIFR as the minimal region necessary for signaling. The signaling reaction appears to involve a cooperativity between the first 70-amino-acid region containing the two sequence motifs conserved among hematopoietin receptors (box 1 and box 2) and a critical sequence between residues 141 and 150 (box 3). Analogous analyses of the cytoplasmic domains of G-CSFR and gpl30 indicated similar arrangements of functional domains in these receptor subunits and the requirement of a box 3-related motif for signaling.

Importance of the Membrane-Proximal Extracellular Domains for Activation of the Signal Transducer Glycoprotein 130

“…Our assumption is that in the D5-GCSFR chimera a kind of disturbance has been introduced that leads to ligand-independent interaction of these proposed dimer interfaces. This idea is supported by the fact that chimeric receptors consisting of the ectodomain of the G-CSFR fused to the transmembrane and cytoplasmic parts of gp130 are only activated in response to G-CSF (35). Thus, D5 of G-CSFR in the context of the complete G-CSFR ectodomain does not lead to ligand-independent activation of gp130.…”

Leptin Receptor (OB-R) Signaling

“…Among members of the class I cytokine receptor family, signaling by the G-CSFR is predicted to be initiated by ligandinduced receptor homodimerization (23,24). Since chimeric receptor complexes have proven quite productive for the analysis of the mechanism of cytokine receptor activation (15,29,30), we constructed OB-R/G-CSFR and G-CSFR/OB-R chimeras as a means to analyze the mechanism of OB-R signaling. We find that the G-CSFR/OB-R chimera can strongly activate transcription of both the IL-6RE-and HRRE-CAT reporter constructs (Fig.…”

Modulation of hepatic acute phase gene expression by epidermal growth factor and src protein tyrosine kinases in murine and human hepatic cells

“…The following vectors have been described: FJ vector, containing avian c-Src 45 (provided by Dr. J.V. Jung, Harvard Medical School, Southborough, MA), pSV containing human EGFR, 46 pDC containing the human chimeric receptor granulocyte colony stimulatory factor receptor (G-CSFR)-gp130-FLAG or G-CSFR-gp130(Y2F)-FLAG (containing the mutated SH-2 domain containing protein tyrosine phosphatase (SHP)-2 binding site in which tyrosine 759 is substituted by phenylalanine, ϭY2F) 47,48 and pIE-MUP containing the murine major urinary protein (MUP) gene. 49 The expression vector for the constitutively active, double mutant MEK-1 (S218D/ S222D) with hematoglutinin antigen tag was purchased from Upstate Biotechnology, Lake Placid, NY.…”

“…In standard transfection protocols, a 1-mL mixture contained 10 to 15 µg of CAT reporter gene construct, 0 to 5 µg of kinase expression vector, 0 to 3 µg of STAT expression vector, 0 to 5 µg of expression vector for receptor subunits, and 1 µg of pIE-MUP as an internal marker. 47,49,60 Empty expression vector was added to bring the DNA to the identical concentration within the experimental series. From the mixture, aliquots representing 1/10 volume of the culture medium were added to HepG2 cell cultures.…”