Cloning and expression of the cDNA for the murine interferon gamma receptor.

Abstract

A murine interferon y (IFN-y) receptor cDNA was isolated by screening a murine T-cell hybridoma library prepared in AgtlO with probes prepared from a human IFN-y receptor cDNA. The 2.1-kilobase (kb) cDNA encoded a serine-rich polypeptide of 477 amino acids that was 52% identical to the human protein. Interferon y (IFN-y) is a potent immunomodulatory cytokine produced by activated T cells and natural killer cells (1). IFN-y is perhaps best recognized for its ability to enhance natural, cell-mediated, and humoral immunity by regulating the differentiation, activation and/or function of a wide variety of immune cell types, such as macrophages (2-4) and B cells (5, 6). However, it also induces a number of other biologic responses on cells, such as fibroblasts (7, 8) (19), and although the murine transfectants displayed the capacity to bind human IFN-y, they failed to respond to it. Thus, it remains uncertain whether unresponsiveness was due merely to limited expression of the human IFN-y receptor at the cell surface or to a deficiency of an additional human component needed to form a functionally active receptor. To further investigate this issue, we examined the biologic responsiveness of human cells that expressed murine IFN-y receptors. In the current communication, we report the cloning and sequencing of a murine IFN-y receptor cDNAAfWe also demonstrate that, unlike its human counterpart, the murine IFN-y receptor cDNA can be stably expressed at high levels in human cells. However, despite expression of 530,000 murine IFN-y receptors per cell, the human cells respond only to the homologous human ligand and not to the murine ligand. These results thus support the concept that additional species-specific components are necessary to form a functionally active IFN-y receptor. MATERIALS AND METHODSReagents. The murine and human IFN-y preparations used in these studies were supplied as highly purified recombinant proteins derived from Escherichia coli and expressed specific antiviral activities of 1 and 3 x 107 units/mg, respectively. The proteins were radioiodinated with Bolton-Hunter reagent (ICN), as described (16). Polyvalent rabbit antirecombinant murine IFN-y (21) was affinity-purified by chromatography on murine IFN-y-Sepharose columns. Purified GR20, a rat monoclonal antibody (mAb) specific for the murine IFN-y receptor (17) was provided

Preassembly and ligand-induced restructuring of the chains of the IFN-γ receptor complex: the roles of Jak kinases, Stat1 and the receptor chains

“…These direct interactions were disrupted by various alterations to the receptor complex described npg with both IFN-γR2 and IFN-γR1 chains. The interaction of IFN-γ with IFN-γR1 is species specific [30,33,34]. Thus, we believe that the overall species-specificity of murine and human IFN-γ for their receptors operates at two levels: binding of IFN-γ to IFN-γR1, and association of IFN-γR2 with IFN-γR1 and IFN-γR2 chains through their extracellular domains.…”

The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.

“…The expression plasmid pRK-M-,yR encoding the mIFN--yR cDNA has been described elsewhere (23). A 1,469-bp fragment, containing the entire coding region of the hIFN--yR cDNA, was inserted into the mammalian cell expression vector pRK5, creating plasmid pRK-H--yR. pRK-HHM--yR encodes a receptor with hIFN--yR sequences for the EC and TM domains and the first four amino acids (Lys-Lys-Ile-Asn) of the IC domain and mIFN--yR sequences for the remainder of the IC domain.…”

“…Human and murine IFN-y share 40%o amino acid sequence identity (22) and exhibit species specificity with respect to receptor binding (15,52) and biological activity (14,22). Human and murine IFN--y receptor (hIFN--yR and mIFN--yR, respectively) cDNAs have been cloned and expressed (2,12,23,25,32,39). The mature hIFN-,yR protein consists of 472 amino acids and shares 52% amino acid sequence identity with the mIFN-yR.…”

“…Human and murine IFN-y share 40%o amino acid sequence identity (22) and exhibit species specificity with respect to receptor binding (15, 52) and biological activity (14,22). Human and murine IFN--y receptor (hIFN--yR and mIFN--yR, respectively) cDNAs have been cloned and expressed (2,12,23,25,32,39 affinity to the hIFN-yR but no biological response is elicited. The hIFN-yR as expressed in murine somatic cell hybrids and transfected mouse cells has binding properties similar to those of the hIFN--yR on human cells (1,7,36,42).…”

Cytokine regulation of expression of class I MHC antigens

“…Both murine and human IFN-γ receptors have been cloned and expressed (Auget et al, 1988;Gray et al, 1989;Hemmi et al, 1989;Kumar et al, 1989;Munru et al, 1989). IFN-γ receptors are 90 kDa glycoproteins and are present on nearly all cell types.…”