Immunohistochemical colocalization of G protein alpha subunits and 5-HT in the rectal gland of the cartilaginous fish Scyliorhinus canicula.

Serotonin [5-hydroxytryptamine (5-HT)] is an important neuromodulator involved in a wide range of physiological functions. The effects of serotonin are mediated by an extended family of receptors coupled to multiple heterotrimeric G-proteins, associated with cellular membrane. G proteins connect receptors to effectors and thus trigger intracellular signaling pathways. These cellular processes several regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis. Generally, elasmobranch fish dwell a hypersaline environment and utilize a specialized extrarenal salt secreting organ, the rectal gland, to face ionic homeostasis. In this study in addition to the morphological, histochemical and immunohistochemical description of the Scyliorhinus canicula rectal gland, for the first time, the presence of serotonin (5-HT), and distribution of different types of G protein alpha subunits (Gα o, Gα q/11, and Gα s/olf) has been investigated in the rectal gland epithelium by confocal immunofluorescence techniques. Colocalization G proteins and 5-HT in the secretory epithelium of the gland suggests serotonin acts as a hormone and involves G proteins in an autocrine-paracrine control of rectal gland homeostasis.

Identification of heterotrimeric G protein α and ß subunits in rice.

Like those in mammals, heterotrimeric G protein complexes have been implicated in signal transduction pathways in plants; however, the subunits themselves have not been isolated. In this study, the rice heterotrimeric G protein subunits α (Gα) and ß (Gß) were purified by affinity chromatography using anti-Gα and -Gß antibodies and SDS-PAGE. Six and seven peptides, respectively, were identified by mass spectrometry and identified as the translation products of the Gα gene RGA1 and Gß gene RGB1. During purification, the subunits dissociated easily from the G protein complex.

Involvement of Gα(olf)-expressing neurons in the vomeronasal system of Bufo japonicus.

Most terrestrial vertebrates possess anatomically distinct olfactory organs: the olfactory epithelium (OE) and the vomeronasal organ (VNO). In rodents, olfactory receptors coupled to Gα(olf) are expressed in the OE, whereas vomeronasal receptors type 1 (V1R) and vomeronasal receptors type 2 (V2R), coupled to Gα(i2) and Gα(o) , respectively, are expressed in the VNO. These receptors and G proteins are thought to play important roles in olfactory perception. However, we previously reported that only V2R and Gα(o) expression is detected in the Xenopus laevis VNO. As X. laevis spends its entire life in water, we considered that expression of limited types of chemosensory machinery in the VNO might be due to adaptation of the VNO to aquatic life. Thus, we analyzed the expression of G proteins in the VNO and the accessory olfactory bulb (AOB) of the adult Japanese toad, Bufo japonicus, because this species is well adapted to a terrestrial life. By using immunohistochemical analysis in combination with in situ hybridization and DiI labeling, we found that B. japonicus Gα(olf) and Gα(o) were expressed in the apical and middle-to-basal layer of the vomeronasal neuroepithelium, and that the axons of these Gα(olf) - and Gα(o) -expressing vomeronasal neurons projected to the rostral and caudal accessory olfactory bulb, respectively. These results strongly suggest that both the Gα(olf) - and Gα(o) -mediated signal transduction pathways function in the B. japonicus VNO. The expression of Gα(olf) in the B. japonicus VNO may correlate with the detection of airborne chemical cues and with a terrestrial life.

Dynamic mass redistribution as a means to measure and differentiate signaling via opioid and cannabinoid receptors.

Classically, G protein-coupled receptor activation by a ligand has been viewed as producing a defined response such as activation of a G protein, activation or inhibition of adenylyl cyclase, or stimulation of phospholipase C and/or alteration in calcium flux. Newer concepts of ligand-directed signaling recognize that different ligands, ostensibly acting at the same receptors, may induce different downstream effects, complicating the selection of a screening assay. Dynamic mass redistribution (DMR), a label-free technology that uses light to measure ligand-induced changes in the mass of cells proximate to the biosensor, provides an integrated cellular response comprising multiple pathways and cellular events. Using DMR, signals induced by opioid or cannabinoid agonists in cells transfected with these receptors were blocked by pharmacologically appropriate receptor antagonists as well as by pertussis toxin. Differences among compounds in relative potencies at DMR versus ligand-stimulated GTPγS or receptor binding endpoints, suggesting functional selectivity, were observed. Preliminary evidence indicates that inhibitors of intermediate steps in the cell signaling cascade, such as receptor recycling inhibitors, mitogen-activated protein kinase kinase/p38 mitogen-activated protein kinase inhibitors, or cytoskeletal disruptors, altered or attenuated the cannabinoid-induced response. Notable is the finding that mitogen-activated protein kinase kinase 1/2 inhibitors attenuated signaling induced by the cannabinoid type 2 receptor inverse agonist AM630 but not that stimulated by the agonist CP 55,940. Thus, DMR has the potential to not only identify ligands that activate a given G protein-coupled receptor, but also ascertain the signaling pathways engaged by a specific ligand, making DMR a useful tool in the identification of biased ligands, which may ultimately exhibit improved therapeutic profiles.

Functional expression of the olfactory signaling system in the kidney.

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons.

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.

Consumption of virgin olive oil influences membrane lipid composition and regulates intracellular signaling in elderly adults with type 2 diabetes mellitus.

We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCalpha) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Galphao, Gbeta, and PKCalpha were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Galphas, Gbeta, and PKCalpha in both groups, reduction in Galphai was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCalpha with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.

Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor.

The vertebrate olfactory epithelium provides an excellent model system to study the regulatory mechanisms of neurogenesis and neuronal differentiation due to its unique ability to generate new sensory neurons throughout life. The replacement of olfactory sensory neurons is stimulated when damage occurs in the olfactory epithelium. In this study, transgenic mice, with a transgene containing human diphtheria toxin receptor under the control of the olfactory marker protein promoter (OMP-DTR), were generated in which the mature olfactory sensory neurons could be specifically ablated when exposed to diphtheria toxin. Following diphtheria toxin induced neuronal ablation, we observed increased numbers of newly generated growth associated protein 43 (GAP43)-positive immature olfactory sensory neurons. OMP-positive neurons were continuously produced from the newly generated GAP43-positive cells. The expression of the signal transduction components adenylyl cyclase type III and the G-protein alpha subunit G(alpha olf) was sensitive to diphtheria toxin exposure and their levels decreased dramatically preceding the disappearance of the OMP-positive sensory neurons. These data validate the hypothesis that OMP-DTR mice can be used as a tool to ablate the mature olfactory sensory neurons in a controlled fashion and to study the regulatory mechanisms of the neuronal replacement.

Implications of lipid raft disintegration: enhanced anti-inflammatory macrophage phenotype.

BACKGROUND: Lipid rafts are membrane microdomains characterized by an enriched cholesterol environment and appear to serve as a platform for signaling. Their role within the macrophage during endotoxin exposure is unknown. METHODS: THP-1 cells were subjected to lipopolysaccharide stimulation with or without methyl-beta-cyclodextrin (MbetaCD) pretreatment, a cholesterol depleting agent. Cell surface expression of toll-like receptor-4 (TLR4) and platelet-activating factor receptor (PAFr) was determined by flow cytometry. Membrane receptor components and activation of the mitogen-activated protein kinases (MAPK) was determined from lipid raft and cellular protein by immunoblot. Inflammatory mediator production was determined from harvested supernatants by enzyme-linked immunosorbent assay. RESULTS: Surface expression of TLR4 and PAFr was not affected by MbetaCD. Lipopolysaccharide stimulation led to TLR4 mobilization to lipid rafts, MAPK activation, and inflammatory mediator production. Pretreatment with MbetaCD did not affect TLR4 mobilization to lipid rafts, but did result in lost lipid raft expression of the PAFr coupled G-protein, Galpha1. MbetaCD treatment led to selective attenuation of MAPK activation through ERK 1/2. This dysregulated signaling was associated with attenuated production of tumor necrosis factor-alpha, but increased production of interleukin-10. CONCLUSION: Lipid raft disintegration results in lost expression of Galpha1, dysregulated MAPK signaling, and selective anti-inflammatory mediator production. Therefore, modulation of lipid raft cholesterol content may represent a potential mechanism for regulation of macrophage phenotypic differentiation.

Identification and quantitation of G-protein alpha-subunits.

The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved on receptor activation by ligand. In addition, these methods can be used to probe the specificity of the interaction and to yield information about the stoichiometries involved.

Cellular localization of GFP-tagged alpha subunits.

Heterotrimeric G proteins transmit signals from a wide range of cell surface G protein-coupled receptors (GPCRs) to mediate multiple cellular events. Within the plasma membrane, G proteins interact with GPCRs and effector proteins such as adenylyl cyclase (AC) and phospholipase C (PLC). Plasma membrane subdomains (e.g., lipid rafts and caveolae) may organize and regulate these interactions. G protein subunits have been reported to be in additional cellular regions, such as the Golgi apparatus and the cytoskeleton, and G protein alpha subunits may move within the cell during the activation cycle. Changes in the cellular localization of alpha subunits could be important for interactions with effectors that are not in the plasma membrane and/or could be a means for terminating G protein signaling. However, until recently, the topic of G protein alpha subunit localization under basal and activated conditions has been controversial, partly because of spatial and temporal limitations inherent to procedures like cell fractionation and immunohistochemistry. Green fluorescent protein (GFP)-tagging is a useful way to enable real-time visualization of proteins in living cells. This chapter describes how to produce and visualize functional GFP-tagged alpha subunits and to investigate whether activation affects their subcellular localization.

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue.

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

Immunohistochemistry of the canine vomeronasal organ.

The canine's olfactory acuity is legendary, but neither its main olfactory system nor its vomeronasal system has been described in much detail. We used immunohistochemistry on paraffin-embedded sections of male and female adult dog vomeronasal organ (VNO) to characterize the expression of proteins known to be expressed in the VNO of several other mammals. Basal cell bodies were more apparent in each section than in rodent VNO and expressed immunoreactivity to anticytokeratin and antiepidermal growth factor receptor antibodies. The thin layer of neurone cell bodies in the sensory epithelium and axon fascicles in the lamina propria expressed immunoreactivity to neurone cell adhesion molecule, neurone-specific beta tubulin and protein gene product 9.5. Some neurones expressed growth-associated protein 43 (GAP43): and a number of those also expressed neurone-specific beta tubulin-immunoreactivity. Some axon fascicles were double labelled for those two proteins. The G-protein alpha subunits Gi and Go, involved in the signal transduction pathway, showed immunoreactivity in the sensory cell layer. Our results demonstrate that the canine vomeronasal organ contains a population of cells that expresses several neuronal markers. Furthermore, GAP43 immunoreactivity suggests that the sensory epithelium is neurogenic in adult dogs.