Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.

Irreversible and bivalent ligands for selected G-protein coupled receptors

Derivatives of pharmaceutical compounds that could potentially bind irreversibly to a hypertension controlling receptor were synthesised to provide tools for studying this receptor. Also compounds that simultaneously activate two cardiovascular receptors with opposing cellular mechanisms were synthesised. This resulted in a desired partial reduction in the activity of one receptor.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT_{1 ha}. The 5-HT_{1 ha} shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT_{1 ha} is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT_{1 ha} belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT_{1 ha}  mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT_{1 ha} specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT_{1 ha} immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

Is fat the sixth taste primary? Evidence and implications

Taste is the chemical sense responsible for the detection of non-volatile chemicals in potential foods. For fat to be considered as one of the taste primaries in humans, certain criteria must be met including: class of affective stimuli; receptors specific for the class of stimuli on taste bud cells (TBC); afferent fibers from TBC to taste processing regions of the brain; perception independent of other taste qualities; and downstream physiological effects. The breakdown products of the macronutrients carbohydrates (sugars) and protein (amino acids) are responsible for activation of sweet and umami tastes respectively. Following the same logic the breakdown products of fat being fatty acids are the likely class of stimuli for fat taste. Indeed, psychophysical studies have confirmed fatty acids of varying chain length and saturation are orally detectable by humans. The most likely fatty acid receptor candidates located on TBC are CD36, G protein-coupled receptor 120. Once the receptors are activated by fatty acids a series of transduction events occurs causing the release of neurotransmitters towards afferent fibers signalling the brain. Whether fatty acids elicit any direct perception independent of other taste qualities is still open to debate with only poorly defined perceptions for fatty acids reported. Others suggest that the fatty acid taste component is at detection threshold only and any perceptions are associated with either aroma or chemesthesis. It has also been established that oral exposure to fat via sham feeding stimulates increases blood triacylglycerol concentrations in humans. Therefore, overall, with the exception of an independent perception, there is consistent emerging evidence that fat is the sixth taste primary. The implications of fatty acid taste go further into health and obesity research with the gustatory detection of fats and their contributions to energy and fat intake receiving increasing attention. There appears to be a coordinated bodily response to fatty acids throughout the alimentary canal; those who are insensitive orally are also insensitive in the gastrointestinal tract and overconsume fatty food and energy. The likely mechanism linking fatty acid taste insensitivity with overweight and obesity is development of satiety after consumption of fatty foods.

Smoothened activates breast cancer stem-like cell and promotes tumorigenesis and metastasis of breast cancer

Smoothened (Smo) is a G protein-coupled receptor protein encoded by the Smo gene of the hedgehog signalling pathway, which is thought to play an important role in maintaining organ patterning, cell differentiation and self-renewal. The possible role of Smo in the process of tumorigenesis and metastasis of breast cancer still remains unclear. The present experiments were to investigate the effect of Smo on activating breast cancer stem-like CD44(+)CD24(-) cells and the tumorigenesis and metastasis of breast cancer. By injected CD44(+)CD24(-) cells (1×10(4)) into the cleared fat pad of NOD/SCID mice, it was observed that CD44(+)CD24(-) cells possess higher tumor-initiating capacity and metastasis properties than equal numbers of non-CD44(+)CD24(-) cells. The mRNA and protein expressions of Smo in CD44(+)CD24(-) cells were higher than those in non-CD44(+)CD24(-) cells, indicating that Smo may play a role in maintaining breast cancer stem cell features. qRT-PCR results revealed that expressions of STAT3, Bcl-2 and cyclinD1 mRNA in MCF-7 cells were decreased after transfected by Smo siRNA. In addition, the expressions of MMP-2 and MMP-9 were downregulated in MCF-7 cells after Smo expression was inhibited. Smo inhibition may be a possible therapeutic target that potentially suppresses breast tumor formation and development.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses.
MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5.
CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.

Serotonin 1a receptor and associated G-protein activation in schizophrenia and bipolar disorder

Abnormalities in the serotonergic signalling system, including the serotonin 1a receptor, have been implicated in the pathogenesis of schizophrenia and bipolar 1 disorder. However, there is no consensus on whether the density of the serotonin 1a receptor and/or the activity of the G-proteins linking the receptor to the intracellular cascade are altered in these disease states. To address these issues, tissue obtained postmortem from four cortical regions was used to measure [3H] 8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) binding and 8-OH-DPAT-stimulated guanosine 5′-[γ-thio]triphosphate (GTPγS) binding to determine if either parameter is altered in schizophrenia or bipolar I disorder. There was an effect of diagnosis on the level of [3H] 8-OH-DPAT binding that may indicate a global change in the density of serotonin 1a receptors, although this effect did not reach significance in any individual brain region. The activation of serotonin 1a receptors did not differ significantly with diagnoses. However, in the outer cortical layers, there appeared to be a dissociation between the number of receptors available and the extent of ligand-induced GTPγS binding, suggesting considerable receptor reserve. In addition, comparing gender independent of diagnoses, a decrease in the levels of serotonin 1a receptors was observed in the cortex of female subjects. These data indicates that there may be subtle changes in serotonin 1a receptors across the cortex in schizophrenia or bipolar I disorder and suggests a gender discordance in receptor levels.

Heterotrimeric G protein γ subunits provide functional selectivity in Gβγ dimer signaling in arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

Short-chain fatty acids increase expression and secretion of stromal cell-derived factor-1 in mouse and human pre-adipocytes

Objective: Stromal cell-derived factor-1 (SDF-1) is expressed in pre-adipocytes but its role is unknown. We investigated butyrate (a histone deacetylase inhibitor - HDACi) and other short-chain fatty acids (SCFA) in the regulation of SDF-1. We further investigated whether effects of SCFA were signalled through G protein-coupled receptors FFA2 and FFA3. Design and Results: SDF-1 mRNA expression and protein secretion were studied in 3T3-L1 cells and human pre-adipocytes. SDF-1 was abundant, with mRNA and protein levels increased by butyrate. This was replicated with acetate and propionate, but not with trichostatin or valproate. Trichostatin inhibited SDF-1 secretion. Pertussis toxin blocked stimulation by butyrate. The order of potency of SCFA in stimulating SDF-1 (C3 > C4 > C2) is consistent with action through FFA3. Silencing the FFA3 gene abolished butyrate-stimulated SDF-1 expression and secretion. FFA3 was expressed in both pre-adipocytes and adipocytes, while FFA2 was expressed in adipocytes only. SDF-1 expression was low in murine macrophage J774.2 cells, while the SDF-1 receptor CXCR4 was absent from 3T3-L1 cells but abundant in J774.2 macrophages. In human pre-adipocytes, FFA3 was also expressed and SCFA increased SDF-1 secretion. Conclusions: SDF-1 and CXCR4 may mediate the interaction between adipose stromal cells and macrophages. Effects of SCFA are mediated through FFA3, but not histone deacetylase inhibition.

A comparative study of rhodopsin function in the great bowerbird (Ptilonorhynchus nuchalis): spectral tuning and light-activated kinetics

Rhodopsin is the visual pigment responsible for initiating the phototransduction cascade in vertebrate rod photoreceptors. Although well-characterized in a few model systems, comparative studies of rhodopsin function, particularly for non-mammalian vertebrates are comparatively lacking. Bowerbirds are rare among passerines in possessing a key substitution, D83N, at a site that is otherwise highly conserved among G protein-coupled receptors. While this substitution is present in some dim-light adapted vertebrates, often accompanying another unusual substitution, A292S, its functional relevance in birds is uncertain. To investigate functional effects associated with these two substitutions, we use the rhodopsin gene from the great bowerbird (Ptilonorhynchus nuchalis) as a background for site-directed mutagenesis, in vitro expression and functional characterization. We also mutated these sites in two additional rhodopsins that do not naturally possess N83, chicken and bovine, for comparison. Both sites were found to contribute to spectral blue-shifts, but had opposing effects on kinetic rates. Substitutions at site 83 were found to primarily affect the kinetics of light-activated rhodopsin, while substitutions at site 292 had a larger impact on spectral tuning. The contribution of substitutions at site 83 to spectral tuning in particular depended on genetic background, but overall, the effects of substitutions were otherwise surprisingly additive, and the magnitudes of functional shifts were roughly similar across all three genetic backgrounds. By employing a comparative approach with multiple species, our study provides new insight into the joint impact of sites 83 and 292 on rhodopsin structure-function as well as their evolutionary significance for dim-light vision across vertebrates.

G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.