The leptin receptor (OB-R) is a single membrane-spanning protein that mediates the weight regulatory effects of leptin (OB protein). The mutant allele (db) of the OB-R gene encodes a protein with a truncated cytoplasmic domain that is predicted to be functionally inactive. Several mRNA splice variants encoding OB-Rs with different length cytoplasmic domains have been detected in various tissues. Here we demonstrate that the full-length OB-R (predominantly expressed in the hypothalamus), but not a major naturally occurring truncated form or a mutant form found in db/db mice, can mediate activation of signal transducer and activator of transcription (STAT) proteins and stimulate transcription through interleukin 6 responsive gene elements. Reconstitution experiments suggest that, although OB-R mediates intracellular signals with a specificity similar to interleukin 6-type cytokine receptors, signaling appears to be independent of the gpl3O signal transducing component of the interleukin 6-type cytokine receptors.

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.

Transient transfection of expression vectors for various members of the hematopoietin receptor family and STAT proteins into COS-1 cells indicated that each recep-tor was capable of stimulating the DNA binding activity of STAT1, STAT3, and STAT5B. However, gp130 preferentially activated STAT1 and STAT3. Activation of STAT5B differed from that of the other two in that the box 3 sequence motif in the cytoplasmic domain of gp130 was not required. Moreover, STAT5B and STAT3 enhanced gene transcription via separate regulatory elements. This study has identified two potential signal transduction pathways by which hematopoietin receptors, including the interleukin-6 receptor, control transcription of acute phase plasma protein genes in hepatic cells.The transcriptional regulation of acute phase plasma protein genes in hepatic cells by 1 has been correlated with the activation of DNA binding properties of STAT3 by the signaling activity of gp130 (1-5). The suggested function of STAT3 as a transcription factor was supported by the finding that overexpression of STAT3 and its activation by cytokine receptor and Janus kinases resulted in enhanced transcription via IL-6-responsive gene elements (6). However, the model proposing a principal role for STAT3 as a mediator of acute phase response (3, 7) needed to be refined because: 1) gp130-dependent transcription via certain elements, such as the HRRE, was found to be independent of STAT3 (8); and 2) the acute phase response of the liver included activation of not only STAT3 but also of other members of the STAT protein family, such as STAT5B (9). 2 The study of the transcription control mechanisms by specific STAT isoforms has been difficult primarily due to the lack of adequate experimental assay systems. Recently, we have developed techniques to reconstitute the function of hepatic and non-hepatic hematopoietin receptors in transiently transfected hepatoma cells (10). We could define the cytoplasmic domains of the signal transducing receptor subunits required for the induction of transcription through specific regulatory elements (8,11). This cell assay system was used to characterize the specificity of STAT protein activation by hematopoietin receptors. Two distinct signaling pathways were identified: one specified by the box 3-dependent activation of STAT3 and transcriptional stimulation via an IL-6RE, and the other specified by the box 3-independent activation of STAT5B and transcriptional stimulation via HRRE. EXPERIMENTAL PROCEDURESCells-HepG2 and COS-1 cells were cultured as described (10). Hormonal treatments were carried out in serum-free medium containing 1 M dexamethasone alone or with 100 ng/ml recombinant human IL-2 (Cetus Corp.), IL-3 (Sandoz), IL-4, G-CSF (Immunex Corp.), GH (Genentech), IL-6 (Genetics Institute), or ovine PRL (National Hormone and Pituitary Program, lot ATP 10677C).Expression Vectors and CAT Reporter Gene Constructs-Expression vectors for the following receptors have been described previously (10 -12): human IL-3R␣ (13), IL-3R␤ (14), IL-2R␤ ...

Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.

The cytoplasmic receptor sequences required for the transcriptional control via the IL-6 response element (IL-6RE) and the hematopoietin receptor response element (HRRE) in hepatoma cells were defined by transient expression of wild-type and mutant granulocyte-colony stimulating factor receptor-gp130 chimeric receptors. gp130 generated two separate transcriptional signals, one of which was directed to IL-6RE and required an intact box 3 motif, and another, which was directed to HRRE and was box 3-independent. The activation of DNA-binding of STAT3 required the same gp130 domains as the IL-6RE response. A box 3-independent activation of STAT proteins was achieved by overexpression of the kinases JAK2 or TYK2. The increase in the DNA-binding activity of STAT proteins, however, did not result in a corresponding increase in transcription via either IL-6RE or HRRE. The data indicate that activation of the DNA-binding potential of STAT proteins via gp130 is not sufficient to achieve transcriptional up-regulation of specific target genes.