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Detection of proteins immunoreactive against an anti-idiotypic antiserum in RINm5F cells. (a) Immunoreactive proteins were visualized by fluorescence microscopy after addition of anti-idiotypic antiserum and a secondary-antibody-FITC conjugate. (b) Immunoreactive proteins were visualized by Western blotting of RINm5F cell proteins with pre-immune (1 and 2) or anti-idiotypic antiserum (3 and 4). The major bands had molecular weights of 40 – 45 kDa, 75 kDa and 80 kDa as judged by reference to molecular weight markers run in parallel.
Source publication
Efaroxan induces membrane depolarization by interaction with the pore forming subunit of the ATP-sensitive potassium channel, Kir6.2. However, this effect is not responsible for its full secretory activity.
In this study we have used an anti-idiotypic approach to generate antibodies that recognize additional proteins that may be regulated by efarox...
Contexts in source publication
Context 1
... sera from the same animals failed to generate positive signals in the ELISA protocol. Direct con®rmation that the sera were reactive against b-cell proteins was obtained by immunostaining of RINm5F cells growing in culture ( Figure 1). Cells were positively stained upon exposure to the antiidiotypic antisera (Figure 1a) whereas no staining was seen with preimmune serum or if the primary antiserum was omitted (not shown). ...
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... con®rmation that the sera were reactive against b-cell proteins was obtained by immunostaining of RINm5F cells growing in culture ( Figure 1). Cells were positively stained upon exposure to the antiidiotypic antisera (Figure 1a) whereas no staining was seen with preimmune serum or if the primary antiserum was omitted (not shown). Signi®cant staining was observed at the periphery of the cells suggesting that at least one of the immunoreactive proteins may be associated with the cell surface. ...
Context 3
... of the antisera in Western blotting experiments revealed that several dierent immunoreactive molecules are expressed in RINm5F cells (Figure 1b). The immunoreactive bands ranged in size from 40 to 80 kDa but none corresponded in molecular weight to that expected of Kir6.2, a molecule that has been previously implicated as a b-cell efaroxan binding protein ( Monks et al., 1999). ...
Context 4
... does not exclude the possibility that the surface immunoreactivity seen in immuno¯uorescence studies may have derived from the labelling of K ATP channels since it is possible that the antiserum may recognize a native conformation of the protein that is not preserved after denaturing gel electrophoresis. Western blotting of RINm5F cell proteins with preimmune sera did not lead to detection of any immunoreactive proteins ( Figure 1b). ...
Citations
... However, Rap1 is activated transiently by glucose 50 and undergoes carboxymethylation. 18, 51 The third group of small G-proteins consists of Rab2, Rhes and Rem2 which are under-studied, [52][53][54][55] whereas, RalA appears to draw direct regulatory effects in exocytosis. 56 Do you have data about small Gproteins expression in pancreas? ...
Glucose-stimulated insulin secretion (GSIS) involves cross talk between small Gproteins
and their regulating factors. These interactions results in translocation of insulin-laden
granules to the plasma membrane for fusion and insulin release. Vesicular transport and fusion
events are tightly regulated by signals which coordinate between vesicle- and membrane-associated
docking proteins. It is now being accepted that small G-protein, Rac1-mediated Reactive
Oxygen Species (ROS) functions as a second messenger in islet β-cell function. Further,
evidence from multiple laboratories suggests a tonic increase in ROS generation is necessary
for GSIS and fatty acid-induced insulin secretion. On the other hand, Rac1-mediated NADPH
oxidase-activation and subsequent generation of excessive ROS under glucolipotoxic conditions
and cytokines exposure has proven to be detrimental for islet β-cell function. In this review
we overview the normal physiological effects (positive role) and adverse effects (negative
role) of activated small G-protein, Rac1 in pancreatic β-cells.
... However, its major level of expression is within the striatum and olfactory tubercle [8,33]. RHES is also expressed outside of the nervous system in the thyroid and pancreas where it might regulate secretion of thyroid hormone and insulin, respectively [16,35]. It is involved in selected striatal competencies mainly locomotor activity and motor coordination suggesting that its downregulation in hypothyroidism could be responsible only for a subset of symptoms, such as the striatopallidal syndrome typical of neurological cretinism [11,[36][37][38]. ...
Dexras1 and RHES, monomeric G proteins, are members of small GTPase family that are involved in modulation of pathophysiological processes. Dexras1 and RHES levels are modulated by hormones and Dexras1 expression undergoes circadian fluctuations. Both these GTPases are capable of modulating calcium ion channels which in turn can potentially modulate neurosecretion/hormonal release. These two GTPases have been reported to prevent the aberrant cell growth and induce apoptosis in cell lines. Present review focuses on role of these two monomeric GTPases and summarizes their role in pathophysiological processes.
... However, RT-PCR indicated low levels in kidney, thyroid, lung, heart, and testis, but no signal in liver (Spano et al., 2004). Furthermore, it was detected in rat and human pancreatic islets (Chan et al., 2002), as well as at low levels in rat adrenal and stomach (Taylor et al., 2006). Within brain, rhes mRNA has been shown to be localized to several areas in addition to the initially described striatal localization: layers 2/3 and 5 of cerebral cortex, piriform cortex, olfactory tubercle and bulb, subiculum, hippocampus (pyramidal and granular layers), anterior thalamic nucleus, cerebellum (granular layer), inferior colliculus, and nucleus accumbens (Vargiu et al., 2004). ...
... Finally, within cell lines, rhes mRNA was detected in undifferentiated PC12 cells, the rat thyroid cell line FRTL-5, and the mouse ES cell line R1, but not in N2A, C6 glioma, or GT1-7 cells (Spano et al., 2004;Vargiu et al., 2004). Rhes mRNA was amplified by RT-PCR from αand β-pancreatic cell lines (αTC1-9, RINm5F, BRIN-BD11, MIN6, and INS-1) and from AtT20 and CHO-K1 cells (Chan et al., 2002;Taylor et al., 2006). However, we do not detect any endogenous Rhes protein in CHO cells by Western blotting . ...
... In addition to these investigations in brain, regulation of Rhes in pancreas has also been examined, but the results are controversial. Initial studies employed anti-idiotypic antibodies that bound to proteins induced by efaroxan, an imidazoline compound that potentiates glucose-induced insulin secretion, and identified Rhes as being regulated by efaroxan (Chan et al., 2002). It was further suggested that Rhes is involved not only in the ability of efaroxan to induce insulin secretion, but also in the desensitization of this response upon chronic treatment (18 h) to rat islets and BRIN-BD11 cells, as a decrease in rhes mRNA expression correlated with a desensitization of efaroxan-induced insulin secretion (Chan et al., 2002). ...
Rhes, the Ras Homolog Enriched in Striatum, is a GTP-binding protein whose gene was discovered during a screen for mRNAs preferentially expressed in rodent striatum. This 266 amino acid protein is intermediate in size between small Ras-like GTP-binding proteins and α-subunits of heterotrimeric G proteins. It is most closely related to another Ras-like GTP-binding protein termed Dexras1 or AGS1. Although subsequent studies have shown that the rhes gene is expressed in other brain areas in addition to striatum, the striatal expression level is relatively high, and Rhes protein is likely to play a vital role in striatal physiology and pathology. Indeed, it has recently been shown to interact with the Huntingtin protein and play a pivotal role in the selective vulnerability of striatum in Huntington's disease (HD). Not surprisingly, Rhes can interact with multiple proteins to affect striatal physiology at multiple levels. Functional studies have indicated that Rhes plays a role in signaling by striatal G protein-coupled receptors (GPCR), although the details of the mechanism remain to be determined. Rhes has been shown to bind to both α- and β-subunits of heterotrimeric G proteins and to affect signaling by both Gi/o- and Gs/olf-coupled receptors. In this context, Rhes can be classified as a member of the family of accessory proteins to GPCR signaling. With documented effects in dopamine- and opioid-mediated behaviors, an interaction with thyroid hormone systems and a role in HD pathology, Rhes is emerging as an important protein in striatal physiology and pathology.
... Thus, Rap1 would be required in the initial step of cell adhesion , which is then maintained by Rap2 signaling [61]. RasD2/Rhes (ras homolog expressed in striatum) is expressed predominately in the striatum [62] but also in thyroid glands and pancreas β-cells [63]. It is involved in selected stritial functions, mainly locomotor activity and motor coordination [64]. ...
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.
We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target.
Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.
... The same study also demonstrated that rhes is differentially expressed in the NAc, with the major levels of expression in the lateral shell, followed by medial shell, and the core. Rhes is also expressed outside of the nervous system in the thyroid and pancreas glands (Chan et al., 2002), where regulate secretion of thyroid hormone and insulin, respectively. Furthermore, during development, the expression of rhes is low during embryonic and early postnatal stages, but increases progressively and become significantly detectable between postnatal days 10 and 15, and decreases during adulthood (Falk et al., 1999;Harrison, Ruskin & LaHoste, 2006). ...
Aims
Reduced cardiac autophagy, ischemic injury, sympathetic overactivity, and apoptosis all contribute to metabolic syndrome (MetS)-associated cardiovascular risks. NR4A2, an orphan nuclear receptor NR4A family member, induces autophagy while suppressing apoptosis in myocardial infarction. Moxonidine, a sympathoinhibitor imidazoline1 receptor (I1R) agonist, has beneficial metabolic and hemodynamic effects; however, whether autophagy and/o NR4A2 signaling are involved moxonidine's cardiovascular effects via I1R activation, is unknown, and is the aim of this study.
Materials and methods
To induce MetS, rats were fed 3 % salt in their diet and 10 % fructose in their drinking water for 12 weeks. MetS-rats were given either moxonidine (6 mg/kg/day, gavage), efaroxan (I1R antagonist, 0.6 mg/kg/day, i.p), both treatments, or vehicles for the last two weeks. Blood pressure, lipid profile, and glycemic control were evaluated. Histopathological examination, circulating cardiac troponin I (c-TnI), proinflammatory interleukin-6 (IL-6), apoptosis (active caspase-3 and Fas-immunostaining), interstitial fibrosis [transforming growth factor-β1 (TGF-β1) and Mallory's trichrome staining], and extracellular matrix remodeling [matrix metalloproteinase-9 (MMP-9)], were used to assess cardiac pathology. Cardiac NR4A2 and its downstream factor, P53, as well as autophagic flux markers, SQSTM1/p62, LC3, and Beclin-1 were also determined.
Key findings
Moxonidine significantly ameliorated MetS-induced metabolic and hemodynamic derangements and the associated cardiac pathology. Moxonidine restored NR4A2 and P53 myocardial levels and enhanced autophagic flux via modulating LC3, Beclin-1, and SQSTM1/p62. Efaroxan reversed the majority of the moxonidine-induced improvements.
Significance
The current study suggests that autophagy modulation via I1R activation is involved in moxonidine-mediated cardiac beneficial effects in MetS.
The existence of binding sites with high affinity for compounds containing the imidazoline moiety first became evident in the mid 1970's when it was found that the hypotensive response elicited by microinjection of the alpha-2 adrenoceptor agonist, clonidine, into the rat brainstem was not mimicked by norepinephrine .....
Rhes is a small GTPase whose expression is highly enriched in striatum. It shares homology with Ras proteins, but also contains a C-terminal extension, thus suggesting additional functions. Signaling by 7 transmembrane receptors through heterotrimeric G proteins is inhibited by Rhes. However, perhaps the most remarkable feature of this small GTPase described thus far is that it can account for the selective vulnerability of the striatum in Huntington's Disease (HD). HD is an autosomal dominant neurodegenerative disease caused by a poly-glutamine expansion in the protein huntingtin. Despite the presence of huntingtin throughout the brain and the rest of the body, the striatum is selectively degenerated. Recent work shows that Rhes acts as an E3 ligase for attachment of SUMO (small ubiquitin-like modifier). As this post-translational modification decreases the formation of huntingtin aggregates and promotes cell death, this property of Rhes offers an explanation for selective striatal vulnerability in HD. In addition, the sequestering of Rhes through its binding to mutant huntingtin may decrease the ability of Rhes to perform vital physiological functions in the neuron. Thus, as Rhes is an attractive candidate for HD therapy, a thorough understanding of its physiological functions will allow for specific targeting of its pathological functions.
Glucose-stimulated insulin secretion from the islet beta-cell involves a sequence of metabolic events and an interplay between a wide range of signaling pathways leading to the generation of second messengers (e.g., cyclic nucleotides, adenine and guanine nucleotides, soluble lipid messengers) and mobilization of calcium ions. Consequent to the generation of necessary signals, the insulin-laden secretory granules are transported from distal sites to the plasma membrane for fusion and release of their cargo into the circulation. The secretory granule transport underlies precise changes in cytoskeletal architecture involving a well-coordinated cross-talk between various signaling proteins, including small molecular mass GTP-binding proteins (G proteins) and their respective effector proteins. The purpose of this article is to provide an overview of current understanding of the identity of small G proteins (e.g., Cdc42, Rac1, and ARF-6) and their corresponding regulatory factors (e.g., GDP/GTP-exchange factors, GDP-dissociation inhibitors) in the pancreatic beta-cell. Plausible mechanisms underlying regulation of these signaling proteins by insulin secretagogues are also discussed. In addition to their positive modulatory roles, certain small G proteins also contribute to the metabolic dysfunction and demise of the islet beta-cell seen in in vitro and in vivo models of impaired insulin secretion and diabetes. Emerging evidence also suggests significant insulin secretory abnormalities in small G protein knockout animals, further emphasizing vital roles for these proteins in normal health and function of the islet beta-cell. Potential significance of these experimental observations from multiple laboratories and possible avenues for future research in this area of islet research are highlighted.
Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing Galphai and Rhes selectively influencing Galphas. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca(2+) channels (Ca(V)2.2) by selectively altering Galphai-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing Ca(V)2.2 and Galphai- or Galphas-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of Ca(V)2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through Galphai-coupled receptors without reducing channel inhibition through a Galphas-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gbetagamma-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on Ca(V)2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gbetagamma signaling and both attenuate receptor-initiated signaling by the Gbetagamma subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca(2+) influx through Ca(V)2.2 channels under more physiological conditions and thereby influence Ca(2+)-dependent events such as neurosecretion.
