Abstract The IFN-γR complex is composed of two IFN-γR1 and two IFN-γR2 polypeptide chains. Although IFN-γR1 is constitutively expressed on all nucleated cells, IFN-γR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-γR2 expression constructs to follow the molecule’s cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255–256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-γR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Δ2) or alanine substitution (LI255–256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Δ2), but not downstream (257Δ2), of Leu-Ile 255–256 also led to overaccumulation. A truncation within the IFN-γR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-γR2 signaling. We have created a series of novel mutants of IFN-γR2 that have facilitated the identification of intracellular domains that control IFN-γR2 accumulation and IFN-γ responsiveness. In contrast to IFN-γR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-γR2 Leu-Ile 255–256. These IFN-γR2 mutants will allow fine dissection of the role of IFN-γ signaling in immunity.
Normal cardiovascular development and physiology depend in part upon signalling through G-protein-coupled receptors (GPCRs), such as the angiotensin II type 1 (AT(1)) receptor, sphingosine 1-phosphate (S1P) receptors and endothelin-1 (ET-1) receptor. Since regulator of G-protein signalling (RGS) proteins function as GTPase-activating proteins for the G alpha subunit of heterotrimeric G-proteins, these proteins undoubtedly have functional roles in the cardiovascular system. In the present paper, we show that human aorta and heart differentially express RGS1, RGS2, RGS3S (short-form), RGS3L (long-form), PDZ-RGS3 (PDZ domain-containing) and RGS4. The aorta prominently expresses mRNAs for all these RGS proteins except PDZ-RGS3. Various stimuli that are critical for both cardiovascular development and function regulate dynamically the mRNA levels of several of these RGS proteins in primary human aortic smooth muscle cells. Both RGS1 and RGS3 inhibit signalling through the S1P(1) (formerly known as EDG-1), S1P(2) (formerly known as EDG-5) and S1P(3) (formerly known as EDG-3) receptors, whereas RGS2 and RGS4 selectively attenuate S1P(2)-and S1P(3)-receptor signalling respectively. All of the tested RGS proteins inhibit AT(1)-receptor signalling, whereas only RGS3 and, to a lesser extent, RGS4 inhibit ET(A)-receptor signalling. The conspicuous expression of RGS proteins in the cardiovascular system and their selective effects on relevant GPCR-signalling pathways provide additional evidence that they have functional roles in cardiovascular development and physiology.
Abstract Choroideremia is an X-linked, blinding retinal degeneration with progressive loss of photoreceptors, retinal pigment epithelial (RPE) cells, and choriocapillaris. To study the extent to which these layers are disrupted in affected males and female carriers, we performed multimodal adaptive optics imaging to better visualize the in vivo pathogenesis of choroideremia in the living human eye. We demonstrate the presence of subclinical, widespread enlarged RPE cells present in all subjects imaged. In the fovea, the last area to be affected in choroideremia, we found greater disruption to the RPE than to either the photoreceptor or choriocapillaris layers. The unexpected finding of patches of photoreceptors that were fluorescently-labeled, but structurally and functionally normal, suggests that the RPE blood barrier function may be altered in choroideremia. Finally, we introduce a strategy for detecting enlarged cells using conventional ophthalmic imaging instrumentation. These findings establish that there is subclinical polymegathism of RPE cells in choroideremia.
J. Neurochem. (2011) 119 , 544–554. Abstract Gβ5 is a divergent member of the signal‐transducing G protein β subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gβ isoforms, Gβ5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling proteins that contain G protein‐γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gβ5 is an essential stabilizer of such regulator of G protein signaling proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gβ5 in the brain outside the visual system. We show here that mice lacking Gβ5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe‐walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gβ5‐deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gβ5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression.
In this study, we show that a single intranasal dose of a plasmid encoding active transforming growth factor beta1 (pCMV-TGF-beta1) prevents the development of T helper cell type 1 (Th1)-mediated experimental colitis induced by the haptenating reagent, 2,4, 6-trinitrobenzene sulfonic acid (TNBS). In addition, such plasmid administration abrogates TNBS colitis after it has been established, whereas, in contrast, intraperitoneal administration of rTGF-beta1 protein does not have this effect. Intranasal pCMV-TGF-beta1 administration leads to the expression of TGF-beta1 mRNA in the intestinal lamina propria and spleen for 2 wk, as well as the appearance of TGF-beta1-producing T cells and macrophages in these tissues, and is not associated with the appearances of fibrosis. These cells cause marked suppression of interleukin (IL)-12 and interferon (IFN)-gamma production and enhancement of IL-10 production; in addition, they inhibit IL-12 receptor beta2 (IL-12Rbeta2) chain expression. Coadministration of anti-IL-10 at the time of pCMV-TGF-beta1 administration prevents the enhancement of IL-10 production and reverses the suppression of IL-12 but not IFN-gamma secretion. However, anti-IL-10 leads to increased tumor necrosis factor alpha production, especially in established colitis. Taken together, these studies show that TGF-beta1 inhibition of a Th1-mediated colitis is due to: (a) suppression of IL-12 secretion by IL-10 induction and (b) inhibition of IL-12 signaling via downregulation of IL-12Rbeta2 chain expression. In addition, TGF-beta1 may also have an inhibitory effect on IFN-gamma transcription.
Abstract Antigen capture and presentation by the gut dendritic cells (DCs) is a complicated process that often involves a direct sampling of the antigens from the lumen. Peyer’s patches (PP) and colonic patches (CP) contain two distinct population of DCs that are thought to participate in the antigen uptake and transport of antigens from the lumen. One group of the DCs sample the lumen by extending the transepithelial dendrites from the lamina propria. The other group of DCs is localized in the intraepithelial compartment itself. In our study we have shown, by intravital imaging of the colon, that the previously reported population of intraepithelial DCs belongs to the fraction of highly motile CD11c+ cells that circulate between crypts and the T-cell and B-cell zones of the CP. We observed that these CD11c+ cells come from the T-cell zone and make a brief contact with the lumen before retreating. Upon examination of the expression profiles of different chemokines we found that CCL20 is expressed on the apical portion of the colonic patch-associated epithelial cells. Administration of anti-CCL20 antibodies blocked homing of CD11c+ cells to the intraepithelial compartment. Motile CD11c+ cells were also able to uptake fluorescent particles and Salmonella bacteria from the lumen and rapidly transport them to the T- and B-cell zones. Thus intraepithelial DCs might play an important role in antigen uptake and trafficking in the gut lymphoid compartments.
