Obestatin regulates adipocyte function and protects against diet-induced insulin resistance and inflammation.

The metabolic actions of the ghrelin gene-derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high-fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3-L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3-L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3-L1 preadipocytes by increasing phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol-induced lipolysis, promoted AMP-activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin-induced glucose uptake. In HFD-fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

Acylated and unacylated ghrelin attenuate isoproterenol-induced lipolysis in isolated rat visceral adipocytes through activation of phosphoinositide 3-kinase γ and phosphodiesterase 3B.

The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic ß-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser(473). Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.

Somatostatin and somatostatin analogues reduce PDGF-induced endometrial cell proliferation and motility.

BACKGROUND: Endometriosis is characterized by ectopic implantation of endometrial cells, which show increased proliferation and migration. Somatostatin (SST) and its analogues inhibit normal and cancer cell growth and motility through the SST receptors, sst1-5. Cortistatin (CST), which displays high structural and functional homology with SST, binds all ssts, as well as MrgX2. Our objective was to investigate the gene expression of the SST/CST system and to determine the effect of SST and its analogues on platelet-derived growth factor (PDGF)-induced proliferation and motility in telomerase-immortalized human endometrial stromal cell (T HESC) line and in primary endometrial stromal cell (ESCs) isolated from human endometriotic tissues. METHODS: Ectopic endometrial tissues were collected from women (n= 23) undergoing laparoscopic surgery for endometriosis (Stage III/IV). Gene expression was evaluated by real-time PCR, cell motility by wound healing assay, protein expression and ß-actin rearrangement by immunofluorescence, cell proliferation by the Alamar blue assay and ERK1/2 and Akt phosphorylation by western blot. RESULTS: Human endometriotic tissues, primary ESCs and T HESCs expressed SST, CST and ssts. SST, its analogues SOM230 and octreotide, as well as CST, counteracted PDGF-induced proliferation and migration in both ESCs and T HESCs. SST also inhibited vascular endothelial growth factor and metalloprotease-2 mRNA expression, and reduced basal and PDGF-induced ERK1/2 phosphorylation. CONCLUSION: These results indicate that the SST/CST system is expressed in endometriotic tissues and cells. The inhibitory effects of SST and its analogues on PDGF-induced proliferation and motility suggest that these peptides may represent promising tools in the treatment of endometriosis.

Heterodimers of adenylyl cyclases 2 and 5 show enhanced functional responses in the presence of Galpha s.

Recent studies have demonstrated that adenylyl cyclase isoforms can form both homo- and heterodimers and that this may be the basic functional unit of these enzymes (see Cooper, D.M.F. and Crossthwaite, A.J. (2006) Trends. Pharmacol. Sci. 8:426-431). Here, we show that adenylyl cyclases 2 and 5 can form a functional heterodimeric complex in HEK293 cells using a combination of BRET, confocal imaging, co-immunoprecipitation and assays of adenylyl cyclase activity. The AC2/5 complex is formed constitutively and is stable in the presence of receptor or forskolin stimulation. The complex formed by AC2/5 is also much more sensitive to the presence of Galpha(s) and forskolin than either of the parent AC isoforms themselves. Finally, we also show that this complex can be detected in native tissues as AC2 and AC5 were localized to the same structures in adult mouse ventricular myocytes and neonatal mouse cardiac fibroblasts and could be co-immunoprecipitated from lysates of mouse heart.

The Distinct Roles of CXCR3 Variants and Their Ligands in the Tumor Microenvironment.

First thought to orchestrate exclusively leukocyte trafficking, chemokines are now acknowledged for their multiple roles in the regulation of cell proliferation, differentiation, and survival. Dysregulation of their normal functions contributes to various pathologies, including inflammatory diseases and cancer. The two chemokine receptor 3 variants CXCR3-A and CXCR3-B, together with their cognate chemokines (CXCL11, CXCL10, CXCL9, CXCL4, and CXCL4L1), are involved in the control but also in the development of many tumors. CXCR3-A drives the infiltration of leukocytes to the tumor bed to modulate tumor progression (paracrine axis). Conversely, tumor-driven changes in the expression of the CXCR3 variants and their ligands promote cancer progression (autocrine axis). This review summarizes the anti- and pro-tumoral activities of the CXCR3 variants and their associated chemokines with a focus on the understanding of their distinct biological roles in the tumor microenvironment.

Signalling complexes associated with adenylyl cyclase II are assembled during their biosynthesis.

We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta2-adrenergic receptors and heterotrimeric G proteins as part of a pre-assembled signalling complex. In this study, we further show that AC interacts with these proteins before it is targetted to the cell surface. Using a combination of approaches including bioluminescence resonance energy transfer (BRET) in concert with subcellular fractionation, we show that ACII and beta2AR initially interact in the ER. Further, dominant-negative Rab1 and Sar1 GTPases which block anterograde trafficking out of the ER have no effect on either ACII/receptor or ACII/Gbetagamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Galpha subunits in ACII signalling complexes suggesting it assembles into the complex at a slightly later stage. Thus, like Kir3.1 inwardly rectifying potassium channels, signalosomes containing ACII are formed during their biosynthesis and not in response to agonist at the cell surface.

Heterooligomerization of human dopamine receptor 2 and somatostatin receptor 2 Co-immunoprecipitation and fluorescence resonance energy transfer analysis.

Somatostatin and dopamine receptors are well expressed and co-localized in several brain regions, suggesting the possibility of functional interactions. In the present study we used a combination of pharmacological, biochemical and photobleaching fluorescence resonance energy transfer (pbFRET) to determine the functional interactions between human somatostatin receptor 2 (hSSTR2) and human dopamine receptor 2 (hD2R) in both co-transfected CHO-K1 or HEK-293 cells as well as in cultured neuronal cells which express both the receptors endogenously. In monotransfected CHO-K1 or HEK-293 cells, D2R exists as a preformed dimer which is insensitive to agonist or antagonist treatment. In control CHO-K1 cells stably co-transfected with hD2R and hSSTR2, relatively low FRET efficiency and weak expression in co-immunoprecipitate from HEK-293 cells suggest the absence of preformed heterooligomers. However, upon treatment with selective ligands, hD2R and hSSTR2 exhibit heterodimerization. Agonist-induced heterodimerization was accompanied by increased affinity for dopamine and augmented hD2R signalling as well as prolonged hSSTR2 internalization. In contrast, cultured striatal neurons display constitutive heterodimerization between D2R and SSTR2, which were agonist-independent. However, heterodimerization in neurons was completely abolished in the presence of the D2R antagonist eticlopride. These findings suggest that hD2R and hSSTR2 operate as functional heterodimers modulated by ligands in situ, which may prove to be a useful model in designing new therapeutic drugs.

Heterogeneity of ghrelin/growth hormone secretagogue receptors. Toward the understanding of the molecular identity of novel ghrelin/GHS receptors.

Ghrelin is a gastric polypeptide displaying strong GH-releasing activity by activation of the type 1a GH secretagogue receptor (GHS-R1a) located in the hypothalamus-pituitary axis. GHS-R1a is a G-protein-coupled receptor that, upon the binding of ghrelin or synthetic peptidyl and non-peptidyl ghrelin-mimetic agents known as GHS, preferentially couples to G(q), ultimately leading to increased intracellular calcium content. Beside the potent GH-releasing action, ghrelin and GHS influence food intake, gut motility, sleep, memory and behavior, glucose and lipid metabolism, cardiovascular performances, cell proliferation, immunological responses and reproduction. A growing body of evidence suggests that the cloned GHS-R1a alone cannot be the responsible for all these effects. The cloned GHS-R1b splice variant is apparently non-ghrelin/GHS-responsive, despite demonstration of expression in neoplastic tissues responsive to ghrelin not expressing GHS-R1a; GHS-R1a homologues sensitive to ghrelin are capable of interaction with GHS-R1b, forming heterodimeric species. Furthermore, GHS-R1a-deficient mice do not show evident abnormalities in growth and diet-induced obesity, suggesting the involvement of another receptor. Additional evidence of the existence of another receptor is that ghrelin and GHS do not always share the same biological activities and activate a variety of intracellular signalling systems besides G(q). The biological actions on the heart, adipose tissue, pancreas, cancer cells and brain shared by ghrelin and the non-acylated form of ghrelin (des-octanoyl ghrelin), which does not bind GHS-R1a, represent the best evidence for the existence of a still unknown, functionally active binding site for this family of molecules. Finally, located in the heart and blood vessels is the scavenger receptor CD36, involved in the endocytosis of the pro-atherogenic oxidized low-density lipoproteins, which is a pharmacologically and structurally distinct receptor for peptidyl GHS and not for ghrelin. This review highlights the most recently discovered features of GHS-R1a and the emerging evidence for a novel group of receptors that are not of the GHS1a type; these appear involved in the transduction of the multiple levels of information provided by GHS and ghrelin.

RGS2 interacts with Gs and adenylyl cyclase in living cells.

Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsalpha, or the Gs-coupled beta2-adrenergic receptor (beta2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsalpha-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the beta2AR. Purified RGS2 selectively bound to the third intracellular loop of the beta2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and beta2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.

Functional beta-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes.

OBJECTIVE: We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. METHODS: Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. RESULTS: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. CONCLUSION: These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Human herpesvirus 8-encoded chemokine vCCL2/vMIP-II is an agonist of the atypical chemokine receptor ACKR3/CXCR7.

The atypical chemokine receptor CXCR7/ACKR3 binds two endogenous chemokines, CXCL12 and CXCL11, and is upregulated in many cancers or following infection by several cancer-inducing viruses, including HHV-8. ACKR3 is a ligand-scavenging receptor and does not activate the canonical G protein pathways but was proposed to trigger ß-arrestin-dependent signaling. Here, we identified the human herpesvirus 8-encoded CC chemokine vCCL2/vMIP-II as a third high-affinity ligand for ACKR3. vCCL2 acted as partial ACKR3 agonist, inducing ß-arrestin recruitment to the receptor, subsequent reduction of its surface levels and its delivery to endosomes. In addition, ACKR3 reduced vCCL2-triggered MAP kinase and PI3K/Akt signaling through other chemokine receptors. Our data suggest that ACKR3 acts as a scavenger receptor for vCCL2, regulating its availability and activity toward human receptors, thereby likely controlling its function in HHV-8 infection. Our study provides new insights into the complex crosstalk between viral chemokines and host receptors as well as into the biology of ACKR3, this atypical and still enigmatic receptor.

Obestatin enhances in vitro generation of pancreatic islets through regulation of developmental pathways.

Availability of large amounts of in vitro generated ß-cells may support replacement therapy in diabetes. However, methods to obtain ß-cells from stem/progenitor cells are limited by inefficient endocrine differentiation. We have recently shown that the ghrelin gene product obestatin displays beneficial effects on pancreatic ß-cell survival and function. Obestatin prevents ß-cell apoptosis, preserves ß-cell mass and stimulates insulin secretion in vitro and in vivo, in both normal and diabetic conditions. In the present study, we investigated whether obestatin may promote in vitro ß-cell generation from mouse pancreatic islet-derived precursor cells. Treatment of cultured islets of Langerhans with obestatin (i) enriched cells expressing the mesenchymal/neuronal marker nestin, which is associated with pancreatic precursors; (ii) increased cell survival and reduced apoptosis during precursor selection; (iii) promoted the generation of islet-like cell clusters (ICCs) with increased insulin gene expression and C-peptide secretion. Furthermore, obestatin modulated the expression of fibroblast growth factor receptors (FGFRs), Notch receptors and neurogenin 3 (Ngn3) during islet-derived precursor cell selection and endocrine differentiation. These results indicate that obestatin improves the generation of functional ß-cells/ICCs in vitro, suggesting implications for cell-based replacement therapy in diabetes. Moreover, obestatin may play a role in regulating pathways involved in pancreas development and regeneration.

Neuroendocrine and metabolic activities of ghrelin gene products.

Acylated ghrelin (AG) is a 28 amino acid gastric peptide a natural ligand for the growth hormone secretagogue (GHS) receptor type 1a (GHS-R1a), endowed with GH-secreting and orexigenic properties. Besides, ghrelin exerts several peripheral metabolic actions, including modulation of glucose homeostasis and stimulation of adipogenesis. Notably, AG administration causes hyperglycemia in rodents as in humans. Ghrelin pleiotropy is supported by a widespread expression of the ghrelin gene, of GHS-R1a and other unknown ghrelin binding sites. The existence of alternative receptors for AG, of several natural ligands for GHS-R1a and of acylation-independent ghrelin non-neuroendocrine activities, suggests that there might be a complex 'ghrelin system' not yet completely explored. Moreover, the patho-physiological implications of unacylated ghrelin (UAG), and obestatin (Ob), the other two ghrelin gene-derived peptides, need to be clarified. Within the next few years, we may better understand the 'ghrelin system', where we might envisage clinical applications.

Unraveling the role of the ghrelin gene peptides in the endocrine pancreas.

The ghrelin gene peptides include acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). AG, mainly produced by the stomach, exerts its central and peripheral effects through the GH secretagogue receptor type 1a (GHS-R1a). UAG, although devoid of GHS-R1a-binding affinity, is an active peptide, sharing with AG many effects through an unknown receptor. Ob was discovered as the G-protein-coupled receptor 39 (GPR39) ligand; however, its physiological actions remain unclear. The endocrine pancreas is necessary for glucose homeostasis maintenance. AG, UAG, and Ob are expressed in both human and rodent pancreatic islets from fetal to adult life, and the pancreas is the major source of ghrelin in the perinatal period. GHS-R1a and GPR39 expression has been shown in beta-cells and islets, as well as specific binding sites for AG, UAG, and Ob. Ghrelin colocalizes with glucagon in alpha-islet cells, but is also uniquely expressed in epsilon-islet cells, suggesting a role in islet function and development. Indeed, AG, UAG, and Ob regulate insulin secretion in beta-cells and isolated islets, promote beta-cell proliferation and survival, inhibit beta-cell and human islet cell apoptosis, and modulate the expression of genes that are essential in pancreatic islet cell biology. They even induce beta-cell regeneration and prevent diabetes in streptozotocin-treated neonatal rats. The receptor(s) mediating their effects are not fully characterized, and a signaling crosstalk has been suggested. The present review summarizes the newest findings on AG, UAG, and Ob expression in pancreatic islets and the role of these peptides on beta-cell development, survival, and function.

Unacylated ghrelin rescues endothelial progenitor cell function in individuals with type 2 diabetes.

OBJECTIVE: Acylated ghrelin (AG) is a diabetogenic and orexigenic gastric polypeptide. These properties are not shared by the most abundant circulating form, which is unacylated (UAG). An altered UAG/AG profile together with an impairment of circulating endothelial progenitor cell (EPC) bioavailability were found in diabetes. Based on previous evidence for the beneficial cardiovascular effects of AG and UAG, we investigated their potential to revert diabetes-associated defects. RESEARCH DESIGN AND METHODS: Healthy human subjects, individuals with type 2 diabetes, and ob/ob mice were AG or UAG infused. EPC mobilization in patients and mice was evaluated, and the underlying molecular mechanisms were investigated in bone marrow stromal cells. Recovered EPCs were also evaluated for the activity of senescence regulatory pathways and for NADPH oxidase activation by knocking down p47(phox) and Rac1. Finally, UAG modulation of human EPC vasculogenic potential was investigated in an in vivo mouse model. RESULTS: Neither AG nor UAG had any effect in healthy subjects. However, systemic administration of UAG, but not AG, prevented diabetes-induced EPC damage by modulating the NADPH oxidase regulatory protein Rac1 and improved the vasculogenic potential both in individuals with type 2 diabetes and in ob/ob mice. In addition, unlike AG, UAG facilitated the recovery of bone marrow EPC mobilization. Crucial to EPC mobilization by UAG was the rescue of endothelial NO synthase (eNOS) phosphorylation by Akt, as UAG treatment was ineffective in eNOS knockout mice. Consistently, EPCs expressed specific UAG-binding sites, not recognized by AG. CONCLUSIONS: These data provide the rationale for clinical applications of UAG in pathologic settings where AG fails.

Intracellular trafficking and assembly of specific Kir3 channel/G protein complexes.

We have previously demonstrated that Kir3.1 channels and Gbeta1gamma2 subunits initially interact in the endoplasmic reticulum (ER). To elucidate the role that anterograde protein trafficking pathways may play in the formation of these complexes, we used dominant negative (DN) mutants of the small G proteins Sar 1 and various compartment-specific Rabs which impede anterograde protein trafficking at different steps. Sar 1 H79G and Rab 1 S25N mutants efficiently blocked the plasma membrane trafficking of the Kir3.1/Kir3.4 complex however they did not block the Gbeta1gamma2/Kir3.1 interaction as measured using bioluminescence resonance energy transfer (BRET). This interaction was also insensitive to the presence of DN Rabs 2, 6, 8, and 11. These results confirm that Gbetagamma/Kir3 complexes form early during channel biosynthesis and trafficking. Using a combination of BRET, protein complementation assays and co-immunoprecipitation, we demonstrate that Gbeta1-4 can interact with Kir3.1 in the absence of Kir3.4. Gbeta5 does not directly interact with the channel but can still be co-immunoprecipitated as part of a larger complex. The interaction between Gbeta and Kir3.1 was selectively fostered by co-expression with different Ggamma subunits. When Ggamma1 or Ggamma11 was co-expressed with eGFP-Gbeta3 or eGFP-Gbeta4, the interaction with the effector was lost. Kir3.2 was capable of interacting with Gbeta1-3 and not Gbeta4 or Gbeta5. These interactions were again fostered by co-expression with Ggamma and were also insensitive to DN Sar 1 or Rab 1. Taken together, our data show that these "precocious" channel/G protein interactions are specific and may have implications beyond their basic function in signalling events.

Novel tools for use in bioluminescence resonance energy transfer (BRET) assays.

Recent advances in imaging assays based on bioluminescence resonance energy transfer (BRET) have made it possible to study protein/protein interactions in living cells under physiological conditions. Here we describe protocols for these assays including relevant positive and negative controls, and we also show how they can be combined with protein complementation assays such as bimolecular fluorescence complementation (BiFC) to study three- and four-partner interactions. We also describe a BRET assay that uses SNAP-tagged proteins as a fluorescence acceptor molecule for the bioluminescent donor.

Ghrelin: a metabolic signal affecting the reproductive system.

Ghrelin, an acylated 28 amino acid gastric peptide, was isolated from the stomach as an endogenous ligand for growth hormone (GH) secretagogue receptor in 1999. Circulating ghrelin is mainly produced by specific cells in the stomach's oxyntic glands. Ghrelin potently stimulates GH release and food intake and exhibits diverse effects, including ones on glucose metabolism and on secretion and motility of the gastrointestinal tract. Besides these effects on food intake and energy homeostasis, ghrelin is also involved in controlling reproductive functions, and a role for it as a novel regulator of the hypothalamic-pituitary gonadal axis is clearly emerging. We review recent ghrelin research with emphasis on its roles in the reproductive axis.

Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.

Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.