Molecular Cloning and Characterization of a Novel Mouse Macrophage Gene That Encodes a Nuclear Protein Comprising Polyglutamine Repeats and Interspersing Histidines

Abstract

Simple tandem repeats of the trinucleotide sequence CAG encode homopolymeric stretches of glutamine. Although polyglutamine has been identified in diverse proteins, it is present predominantly in transcription factors. We observed that oncogene-immortalized mouse macrophages express several genes that contain a CAG repeat motif. Therefore, we attempted to clone a novel gene that contains a CAG repeat and is associated with cytokine activation of macrophages. Screening of a mouse macrophage cDNA library with a probe comprising 12 consecutive CAG triplets identified at least one unique clone. The cDNA encodes a protein (named GRP-1 or glutamine repeat protein-1) with 171 amino acids, a calculated molecular mass of 21.6 kDa, and a predicted pI of 10.67. Greater than two-thirds of GRP-1 are only two amino acids, namely glutamine (50%) and histidine (18%). There are four polyglutamine motifs interspersed with histidine-rich regions. There is also a putative nuclear localization signal flanked by sites for possible serine phosphorylation. GRP-1 mRNA was expressed constitutively in some macrophage cell lines and B and T cell lines. Interferon-␥ or lipopolysaccharide augmented GRP-1 mRNA expression in the mouse macrophage cell line ANA-1. Western blot analyses using an antipeptide serum revealed that GRP-1 was localized in the nucleus of ANA-1 macrophages and transfected 3T3 fibroblasts. Overexpression of GRP-1 decreased Sp1-driven chloramphenicol acetyltransferase gene expression in transient cotransfection experiments. Because polyglutamine motifs can cause protein oligomerization and can function as transcriptional activation domains, we suggest that GRP-1 may be a transcription factor associated with interferon-␥-or lipopolysaccharide-induced activation of macrophages.Macrophages play a major role in the immune system through numerous activities expressed either constitutively or after exposure to cytokines, bacterial lipopolysaccharide (LPS) 1 or other agents either alone or in combination (1). For example, macrophages affect T-cell activation by processing and presenting antigens, and they promote inflammatory reactions by secreting various cytokines. In addition, activated macrophages exert potent antibacterial, antiviral, and antitumor functions.To avoid the difficulties associated with isolating large homogeneous populations of macrophages for studying such functions, several oncogene-immortalized mouse macrophage cell lines were generated that emulate normal tissue macrophages (2-4). Although these and other macrophage populations do not synthesize and secrete IL-2, Northern blot analyses revealed the expression of several genes in oncogene-immortalized macrophages that were at least partially homologous with mouse IL-2 cDNA. The expression of at least one of these genes was regulated by IFN-␥ and/or LPS. Subsequent experiments showed that these macrophage genes were homologous with sequences contained in exon I of the mouse IL-2 gene. 2 This region is particularly noteworthy because it contains a tandem repea...

Temperature Entrainment of Drosophila's Circadian Clock Involves the Gene nocte and Signaling from Peripheral Sensory Tissues to the Brain

“…Although Nocte has no apparent DNA binding domain, the presence of poly-Q and poly-A stretches also suggests that Nocte may function as transcription cofactor (Riley and Orr, 2006), which is further supported by a stretch of 268 amino acids showing weak homology to the mammalian Glutamine Rich Protein 1 (GRP-1) ( Figure 3C; Cox et al, 1996). Comparison with available genome sequences revealed that nocte is distributed among insects (i.e., not only Drosophilidae; Figure 3C), but no obvious vertebrate homolog was identified (Experimental Procedures).…”

Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation

“…Homopolymeric tracts of glutamine are not uncommon and have been found in a number of mammalian transcription factors (5,6,11,12,25). Moreover, recent studies have shown that homopolymeric stretches of glutamines are also found in other serine/threonine protein kinases which have a high degree of similarity with C. trifolii TB3.…”

Impaired maturation of myeloid progenitors in mice lacking novel Polycomb group protein MBT-1

“…Interestingly, the mouse homolog of MBT-1 harbors a unique glutamine-rich domain (poly-Q), which is absent in human MBT-1. This domain probably strengthens protein-protein interactions, resulting in the polymerization/ aggregation of nuclear molecules, as in macrophage glutamine repeat protein-1 (GRP-1) (Cox et al, 1996). Other components are, however, highly conserved in human and mouse clones ( Figure 1A).…”