No abstract

IFN‐γ is a key immunoregulatory cytokine that plays a predominant role in innate immunity. By employing PCR‐Select to search for genes differentially expressed in IFN‐γ/TNF‐α stimulated macrophages, we identified a novel IFN‐γ‐induced transcript designated PUMA‐G (protein up‐regulated in macrophages by IFN‐γ). PUMA‐G codes for a protein with seven transmembrane helices, a feature commonly shared with the G protein‐coupled receptor superfamily (GPCR). The PUMA‐G protein is most similar to the human orphan GPCR HM74 (73 % identity) and GPR31 (30 % identity). PUMA‐G mRNA was readily induced in macrophages after stimulation with IFN‐γ, LPS, polyIC and CpG oligonucleotides. In vivo PUMA‐G was up‐regulated in mice suffering from microbial sepsisor from Listeria monocytogenes infection. Characterization of the genomic locus revealed an intronless PUMA‐G open reading frame. Genomic Southern blot analysis indicates that PUMA‐G is a single‐copy gene. PUMA‐G maps to mouse chromosome 5F. Confocal microscopy of transiently transfected 264.7 RAW macrophages and 293T cells with a PUMA‐G‐EGFP fusion construct showed predominant fluorescence at the cell surface, suggesting a localization at the cell membrane. Taken together, our data indicate that PUMA‐G is a new inducible representative of GPCR, with potential importance in macrophage functions.

IFN-γ induces a number of cellular programs functional in innate and adaptive resistance to infectious pathogens. It has recently become clear that the complete cellular response to IFN-γ is extraordinarily complex, with >500 genes (i.e., ∼0.5% of the genome) activated. We made suppression-subtractive hybridization differential libraries from IFN-γ-stimulated primary mouse embryonic fibroblasts and from a mouse macrophage cell line, ANA-1, in each case with reference to unstimulated cells. Of ∼250 clones sequenced at random from the two libraries, >35% were representatives of one or the other of two small unrelated families of GTPases, the 65-kDa and 47-kDa families. These families dominate the IFN-γ-induced response in both cell types. We report here the full-length sequences of one new 65-kDa and two new 47-kDa family members. The 65-kDa family members are under transcriptional control of IRF-1, whereas the 47-kDa family members are inducible in embryonic fibroblasts from IRF-1−/− mice. Members of both GTPase families are strongly up-regulated in livers of wild-type mice infected with the pathogenic bacterium, Listeria monocytogenes, but not in IFN-γR0/0 mice. These GTPases appear to be dedicated to the IFN-γ response, since resting levels are negligible and since neither family shows any significant relationship to any other described family of GTPases. Understanding the role of these GTPases in IFN-γ-mediated resistance against pathogens is the task for the future.