Dual Oncostatin M (OSM) Receptors: CLONING AND CHARACTERIZATION OF AN ALTERNATIVE SIGNALING SUBUNIT CONFERRING OSM-SPECIFIC RECEPTOR ACTIVATION

Bruce Mosley,Cyr De Imus,Della Friend,Norman Boiani,Bettina Thoma,Linda S Park,David Cosman

doi:10.1074/jbc.271.51.32635

Bruce Mosley, Cyr De Imus + Show 5 more

Open Access

https://doi.org/10.1074/jbc.271.51.32635

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Abstract

Oncostatin M (OSM) is a cytokine whose structural and functional features are similar to other members of the interleukin (IL)-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF), granulocyte colonystimulating factor, ciliary neurotrophic factor, and cardiotrophin-1), many of which utilize gp130 as a common receptor subunit. A biologically active OSM receptor has been previously described that consists of a heterodimer of leukemia inhibitory factor receptor (LIFR) and gp130. This LIFR.gp130 complex is also a functional receptor for LIF. We have cloned and characterized an alternative subunit (OSMRbeta) for an OSM receptor complex (a heterodimer of gp130 and OSMRbeta) that is activated by OSM but not by LIF. The signaling capability of specific receptor subunit combinations was analyzed by independent assays measuring cell proliferation or induction of acute phase protein synthesis. Our results demonstrate that both LIF and OSM cause tyrosine phosphorylation and activation of the gp130.LIFR combination, but the gp130.OSMRbeta complex is activated by OSM only. OSM-induced cellular responses, initiated through low affinity binding to gp130, are mediated by two heterodimeric receptor complexes that utilize alternative signal transducing subunits that confer different cytokine specificities to the receptor complex.

Highlights

Oncostatin M (OSM) is a cytokine whose structural and functional features are similar to other members of the interleukin (IL)-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF), granulocyte colonystimulating factor, ciliary neurotrophic factor, and cardiotrophin-1), many of which utilize gp130 as a common receptor subunit
A subset of hematopoietin receptors which share higher overall homology are distinct from other hematopoietin receptors in having three fibronectin type III repeats separating the hematopoietin domain from the transmembrane domain (LIFR contains a second hematopoietin domain at the amino terminus)
OSM signaling on responsive cells is mediated by binding to two distinct signaling receptor complexes identified as type I and type II OSM receptors

Summary

EXPERIMENTAL PROCEDURES

Generation of PCR Library—The pool of sense orientation degenerate oligonucleotides TT(T/G)(C/A)(G/A)(G/T)(A/G)T(T/A)CG(C/G/T)(T/ A)G(T/C) and antisense oligonucleotides (A/G)CTCCA(G/T)T(C/T)(G/ A)CTCCA were used to perform PCR amplification utilizing human genomic DNA (Clontech Laboratories, Inc., Palo Alto, CA) as template. Proliferation Assay—Generation of BAF-B03 cell lines expressing gp130 and gp130/LIFR has been described [31]. To create a cell line expressing gp130 and OSMR␤, full-length OSMR␤ cDNA was subcloned into the expression vector pDC411 derived from the previously described pDC409 vector [32], modified to contain the hygromycin resistance gene, and electroporated into BAF-gp130 cells. DNA encoding the hematopoietin receptor domains of gp130, LIFR, and OSMR␤ was each fused to the Fc region of human IgG1 (gp130-Fc, LIFR-Fc, and OSMR␤-Fc, respectively) and expressed in CV-1/EBNA cells as described [38]. Binding studies with BAF-BO3 cell lines expressing gp130 and gp130/OSMR␤ were performed as described previously [31]. Blots were hybridized overnight with 32P-labeled riboprobes from the entire coding region and washed using high stringency conditions (see above)

RESULTS

DISCUSSION

Pancreatic tumor