PMID: 23727611;PMCID: PMC3717261;Abstract:
We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48h post-feeding. © 2013 Elsevier Ltd.
Glucocorticoids, a class of steroid hormones, associate specifically with intracellular receptors, facilitating a conformational change that converts the receptor in vitro to a DNA-binding protein and in vivo to a nuclear species that activates a class of transcriptional enhancers termed glucocorticoid response elements (GREs). The DNA sequences recognized specifically by the hormone-receptor complex correspond directly to those required for GRE enhancement. The structural transition that accompanies steroid binding, 'receptor transformation', has been monitored by changes in receptor chromatographic properties, accessibility to monoclonal antibodies, association with other receptor subunits or with heterologous proteins, and aqueous two-phase partition coefficient. However, the significance of the structural change for the biological activity of the receptor is not understood. We have used cloned rat glucocorticoid-receptor coding sequences to produce and characterize a novel class of receptor mutants that elicit GRE enhancer function in transfected cells even in the absence of hormone. The constitutive activity of those receptor derivatives, together with mapping studies that distinguish between the DNA- and hormone-binding domains of the receptor, imply that the conformational change corresponding to receptor transformation may simply unmask pre-existing functional domains for DNA binding, enhancer activation, or both.
We developed a molecular genetic model to investigate glucocorticoid receptor (GR) signaling in human bronchial epithelial cells in response to the therapeutic steroid budesonide. Based on a genetic selection scheme using the human Chago K1 cell line and integrated copies of a glucocorticoid-responsive herpes simplex virus thymidine kinase gene and a green fluorescent protein gene, we isolated five Chago K1 variants that grew in media containing budesonide and ganciclovir. Three spontaneous budesonide-resistant subclones were found to express low levels of GR, whereas two mutants isolated from ethylmethane sulfonate-treated cultures contained normal levels of GR protein. Analysis of the GR coding sequence in the budesonide-resistant subclone Ch-BdE5 identified a novel Val to Met mutation at amino acid position 575 (GRV575M) which caused an 80% decrease in transcriptional regulatory functions with only a minimal effect on ligand binding activity. Homology modeling of the GR structure in this region of the hormone binding domain and molecular dynamic simulations suggested that the GRV575M mutation would have a decreased affinity for the LXXLL motif of p160 coactivators. To test this prediction, we performed transactivation and glutathione-S-transferase pull-down assays using the p160 coactivator glucocorticoid interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 and found that GR V575M transcriptional activity was not enhanced by GRIP1 in transfected cells nor was it able to bind GRIP1 in vitro. Identification of the novel GRV575M variant in human bronchial epithelial cells using a molecular genetic selection scheme suggests that functional assays performed in relevant cell types could identify subtle defects in GR signaling that contribute to reduced steroid sensitivities in vivo.
Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.
Androgen receptor (AR) and glucocorticoid receptor (GR) belong to the same subfamily of steroid/nuclear receptors and have been shown to bind qualitatively to the same hormone response element (HRE) DNA sequences. Despite this similarity in target gene recognition, AR and GR have differential affects on the transcriptional regulation of genes containing both simple and complex HRE control regions. Using HREs from the mouse mammary tumor virus (MMTV), tyrosine aminotransferase (TAT), prostatein (C3) or sexlimited protein (SLP) genes, linked to the thymidine kinase promoter, we found receptor-selective differences in the ability of rat AR and rat GR to induce transcription of these various reporter genes. Since AR and GR have a 20% amino acid sequence difference in their DNA binding domains (DBDs), which could result in altered DNA binding affinities, we measured the ability of purified AR and GR DBDs to bind selectively and with high affinity to these HRE sequences in vitro. Gel shift mobility assays showed that the GR DBD had a higher affinity for a consensus HRE than did the AR DBD, and quantitative DNase I footprinting revealed that AR and GR DBDs bound to the MMTV, TAT, C3 and SLP HREs with different affinities. It was found that AR had a dissociation constant (Kd) that was 2-3 times higher than GR on the TAT, C3 and SLP HREs and that the Kd of AR for the C3 and SLP HREs differed by an order of magnitude (43 nM and 460 nM, respectively). Taken together, these data suggest that amino acid differences in the AR and GR DBDs contribute to altered receptor-DNA interactions, however it is likely that non-receptor factors are involved in further modulating receptor-selective DNA binding and transactivation functions. © 1995.