Endothelin-converting enzyme-1 regulates glucagon-like peptide-1 receptor signalling and resensitisation.

Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) is secreted from intestinal L-cells and mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic ß-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving ß-cell mass. These effects are mediated by the GLP-1 receptor (GLP-1R), which is a therapeutic target in type 2 diabetes. Although agonism at the GLP-1R has been well studied, desensitisation and resensitisation are perhaps less well explored. An understanding of these events is important, particularly in the design and use of novel receptor ligands. Here, using either HEK293 cells expressing the recombinant human GLP-1R or the pancreatic ß-cell line, INS-1E with endogenous expressesion of the GLP-1R, we demonstrate GLP-1R desensitisation and subsequent resensitisation following removal of extracellular GLP-1 7-36 amide. Resensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Resensitisation is also regulated by endothelin-converting enzyme-1 (ECE-1) activity, most likely through proteolysis of GLP-1 in endosomes and the facilitation of GLP-1R dephosphorylation and recycling. Inhibition of ECE-1 activity also increases GLP-1-induced activation of extracellular signal-regulated kinase and generation of cAMP, suggesting processes dependent upon the lifetime of the internalised ligand-receptor complex.

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor.

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1 and NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are biased by the ligand bound and can therefore influence signaling function. Using recombinant cell lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared with NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer lasting if NmS is the ligand. Furthermore, when cells are briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained. However, only NmU responses are potentiated and extended by endothelin-converting enzyme-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction.

Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2).

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Characterization of the migration of lung and blood T cells in response CXCL12 in a three-dimensional matrix.

The ability of T cells to microlocalize within tissues, such as the lung, is crucial for immune surveillance and increased T-cell infiltration is a feature of many inflammatory lung conditions. T-cell migration has mainly been studied in two-dimensional assays. Using three-dimensional collagen gels to mimic the extracellular matrix of lung tissue, we have characterized the migration of T lymphocytes isolated from peripheral blood (PBT) and lung (LT) in response to interleukin-2 (IL-2) and CXCL12. Freshly isolated PBT and LT showed a low degree of migration (blood 4.0 +/- 1.3% and lung 4.1 +/- 1.7%). Twenty-four hours of culture increased the percentage of migrating PBT and LT (blood 17.5 +/- 2.9% and lung 17.7 +/- 3.8%). The IL-2 stimulation modestly increased migration of PBT after 6 days (32.3 +/- 6.0%), but had no effect on the migration of LT (25.5 +/- 3.2%). Twenty-four hours of stimulation with anti-CD3/CD28 caused a small but significant increase in the migration of PBT (to 36.4 +/- 5.8%). In a directional three-dimensional assay, CXCL12 failed to induce migration of fresh PBT or LT. Twenty-four hours of culture, which increased CXCR4 expression of PBT 3.6-fold, significantly increased the migration of PBT in response to CXCL12. Migration of PBT to CXCL12 was blocked by pertussis toxin, but not by the phosphoinositide 3-kinase inhibitor wortmannin. Twenty-four-hour cultured LT did not respond to CXCL12. CD3/CD28-stimulation inhibited CXCL12-mediated migration of PBT. These results suggest that the migration pattern of PBT is distinct from that of LT.

Comparative effects of the endogenous agonist glucagon-like peptide-1 (GLP-1)-(7-36) amide and the small-molecule ago-allosteric agent "compound 2" at the GLP-1 receptor.

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors.

Sigma-1 and sigma-2 receptors are emerging therapeutic targets. Although the molecular identity of the sigma-2 receptor has recently been determined, receptor quantitation has used, and continues to use, the sigma-1 selective agents (+) pentazocine or dextrallorphan to mask the sigma-1 receptor in radioligand binding assays. Here, we have assessed the suitability of currently established saturation and competition binding isotherm assays that are used to quantify parameters of the sigma-2 receptor. We show that whilst the sigma-1 receptor mask (+) pentazocine has low affinity for the sigma-2 receptor (Ki 406 nM), it can effectively compete at this site with [³H] di-O-tolyl guanidine (DTG) at the concentrations frequently used to mask the sigma-1 receptor (100 nM and 1 µM). This competition influences the apparent affinity of DTG and other ligands tested in this system. A more troublesome issue is that DTG can displace (+) pentazocine from the sigma-1 receptor, rendering it partly unmasked. Indeed, commonly used concentrations of (+) pentazocine, 100 nM and 1 µM, allowed 37 and 11% respectively of sigma-1 receptors to be bound by DTG (300 nM), which could result in an overestimation of sigma-2 receptor numbers in assays where sigma-1 receptors are also present. Similarly, modelled data for 1 µM dextrallorphan show that only 71-86% of sigma-1 receptors would be masked in the presence of 300 nM DTG. Therefore, the use of dextrallorphan as a masking agent would also lead to the overestimation of sigma-2 receptors in systems where sigma-1 receptors are present. These data highlight the dangers of using masking agents in radioligand binding studies and we strongly recommend that currently used masking protocols are not used in the study of sigma-2 receptors. In order to overcome these problems, we recommend the use of a cell line apparently devoid of sigma-1 receptors [e.g., MCF7 (ATCC HTB-22)] in the absence of any masking agent when determining the affinity of agents for the sigma-2 receptor. In addition, assessing the relative levels of sigma-1 and sigma-2 receptors can be achieved using [³H] DTG saturation binding followed by two-site analysis of (+) pentazocine competition binding with [³H] DTG.

Full and partial agonists of muscarinic M3 receptors reveal single and oscillatory Ca2+ responses by beta 2-adrenoceptors.

Under physiological circumstances, cellular responses often reflect integration of signaling by two or more different receptors activated coincidentally or sequentially. In addition to heterologous desensitization, there are examples in which receptor activation either reveals or potentiates signaling by a different receptor type, although this is perhaps less well explored. Here, we characterize one such interaction between endogenous receptors in human embryonic kidney 293 cells in which Galpha(q/11)-coupled muscarinic M(3) receptors facilitate Ca(2+) signaling by Galpha(s)-coupled beta(2)-adrenoceptors. Measurement of changes in intracellular [Ca(2+)] demonstrated that noradrenaline released Ca(2+) from thapsigargin-sensitive intracellular stores only during activation of muscarinic receptors. Agonists with low efficacy for muscarinic receptor-mediated Ca(2+) responses facilitated cross-talk more effectively than full agonists. The cross-talk required Galpha(s) and was dependent upon intracellular Ca(2+) release channels, particularly inositol (1,4,5)-trisphosphate receptors. However, beta(2)-adrenoceptor-mediated Ca(2+) release was independent of measurable increases in phospholipase C activity and resistant to inhibitors of protein kinases A and C. Interestingly, single-cell imaging demonstrated that particularly lower concentrations of muscarinic receptor agonists facilitated marked oscillatory Ca(2+) signaling to noradrenaline. Thus, activation of muscarinic M(3) receptors profoundly influences the magnitude and oscillatory behavior of intracellular Ca(2+) signaling by beta(2)-adrenoceptors. Although these receptor subtypes are often coexpressed and mediate contrasting acute physiological effects, altered oscillatory Ca(2+) signaling suggests that cross-talk could influence longer term events through, for example, regulating gene transcription.

Elk-1, CREB, and MKP-1 regulate Egr-1 expression in gonadotropin-releasing hormone stimulated gonadotrophs.

Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin enhances expression of the zinc finger transcription factor Egr-1 in a pituitary gonadotroph cell line. The signaling cascade is blocked by overexpression of MAP kinase phosphatase-1 that dephosphorylates extracellular signal-regulated protein kinase in the nucleus. Chromatin immunoprecipitation experiments revealed that the phosphorylated form of Elk-1, a key regulator of gene transcription driven by serum response element (SRE), binds to the 5'-upstream region of the Egr-1 gene in buserelin-stimulated gonadotrophs. Expression of a dominant-negative mutant of Elk-1 completely blocked Egr-1 expression, indicating that Elk-1 connects the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the Egr-1 gene. GnRH receptor activation additionally induced the phosphorylation of CREB, which in its phosphorylated form bound to the Egr-1 gene. Expression of a dominant-negative mutant of CREB reduced GnRH receptor-induced upregulation of Egr-1 expression, indicating that CREB plays a role in the signaling pathway that regulates Egr-1 expression in gonadotrophs. We further identified the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, and transforming growth factor beta as bona fide target genes of Egr-1 in gonadotrophs. The analysis of gonadotroph cells that express--in addition to GnRH receptors--muscarinic M(3) acetylcholine receptors revealed that the nuclear events connecting GnRH receptors and muscarinic M(3) acetylcholine receptors with the Egr-1 gene are indistinguishable.

Paradoxical behavior of neuromedin U in isolated smooth muscle cells and intact tissue.

Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

Lack of receptor-selective effects of either RGS2, RGS3 or RGS4 on muscarinic M3- and gonadotropin-releasing hormone receptor-mediated signalling through G alpha q/11.

Termination of signalling by G-protein-coupled receptors requires inactivation of the G alpha-subunits of heterotrimeric G-proteins and the re-association of G alpha- and G betagamma-subunits. Inactivation of G alpha-subunits is achieved by the hydrolysis of bound GTP by an intrinsic GTPase activity, which is considerably enhanced by GTPase activating proteins. Regulators of G-protein signalling (RGS) proteins are a large family of GTPase activating proteins, many of which have structures indicating roles beyond GTPase activating protein activity and suggesting that the identity of the RGS protein recruited may also be critical to other aspects of signalling. There is some evidence of selective effects of RGS proteins against different G-protein-coupled receptors coupling to the same signalling pathways and growing evidence of physical interactions between RGS proteins and G-protein-coupled receptors. However, it is unclear as to how common such interactions are and the circumstances under which they are functionally relevant. Here we have examined potential selectivity of RGS2, 3 and 4 against signalling mediated by G alpha q/11-coupled muscarinic M3 receptors and gonadotropin-releasing hormone in an immortalised mouse pituitary cell line. Despite major structural differences between these two receptor types and agonist-dependent phosphorylation of the muscarinic M3- but not gonadotropin-releasing hormone receptor, signalling by both receptors was similarly inhibited by expression of either RGS2 or RGS3, whereas RGS4 has little effect. Thus, at least in these circumstances, RGS protein-dependent inhibition of signalling is not influenced by the nature of the G-protein-coupled receptor through which the signalling is mediated.

Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+.

Alteration in [Ca(2+)](i) (the intracellular concentration of Ca(2+)) is a key regulator of many cellular processes. To allow precise regulation of [Ca(2+)](i) and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca(2+)](i) both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca(2+) from intracellular stores and influence Ca(2+) entry across the plasma membrane. It has been well documented that Ca(2+) signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca(2+) signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.

The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

Small lipidated anti-obesity compounds derived from neuromedin U.

A small library of truncated/lipid-conjugated neuromedin U (NmU) analogs was synthesized and tested in vitro using an intracellular calcium signaling assay. The selected, most active analogs were then tested in vivo, and showed potent anorexigenic effects in a diet-induced obese (DIO) mouse model. The most promising compound, NM4-C16 was effective in a once-weekly-dose regimen. Collectively, our findings suggest that short, lipidated analogs of NmU are suitable leads for the development of novel anti-obesity therapeutics.

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells.

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.

Confocal microscopy: theory and applications for cellular signaling.

The development of confocal microscopy and the commercial availability of confocal microscopes have provided many laboratories with an extremely powerful approach to examine cellular structure and function. Allied with the development of suitable tools, it is now possible to interrogate a wide range of structural and functional aspects on both fixed and live cells. Here we describe the basic principles underlying confocal microscopy and provide methodological accounts of how it can be used to study aspects related particularly (but not exclusively) to the expression, activation, and regulation of signaling by G-protein-coupled receptors. Specifically we provide detailed protocols for examining: the cellular expression and distribution of proteins by immunocytochemistry; cytoplasmic and organelle Ca(2+) signaling using fluorescent indicators; second messenger generation using fluorescently tagged biosensors; and ligand/receptor internalization using fluorescently tagged peptide agonists and receptors.

Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.

Allosteric modulation of the activity of the glucagon-like peptide-1 (GLP-1) metabolite GLP-1 9-36 amide at the GLP-1 receptor.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrients has many physiological effects but particularly enhances glucose-dependent insulin release through the GLP-1 receptor (GLP-1R). GLP-1 7-36 amide, the predominant circulating active form of GLP-1, is rapidly truncated by dipeptidyl peptidase-4 to GLP-1 9-36 amide, which is generally considered inactive. Given its physiological roles, the GLP-1R is targeted for treatment of type 2 diabetes. Recently 'compound 2' has been described as both an agonist and positive allosteric modulator of GLP-1 7-36 amide affinity, but not potency, at the GLP-1R. Importantly, we demonstrated previously that exendin 9-39, generally considered a GLP-1R antagonist, enhances compound 2 efficacy (or vice versa) at the GLP-1R. Given that GLP-1 9-36 amide is the major circulating form of GLP-1 post-prandially and is a low affinity weak partial agonist or antagonist at the GLP-1R, we investigated interaction between this metabolite and compound 2 in a cell line with recombinant expression of the human GLP-1R and the rat insulinoma cell line, INS-1E, with native expression of the GLP-1R. We show compound 2 markedly enhances efficacy and potency of GLP-1 9-36 amide for key cellular responses including AMP generation, Ca(2+) signaling and extracellular signal-regulated kinase. Thus, metabolites of peptide hormones including GLP-1 that are often considered inactive may provide a means of manipulating key aspects of receptor function and a novel therapeutic strategy.

Evidence that Ca2+ within the microdomain of the L-type voltage gated Ca2+ channel activates ERK in MIN6 cells in response to glucagon-like peptide-1.

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic ß-cells potentiating glucose-stimulated insulin secretion and stimulating ß-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca(2+) through L-type voltage gated Ca(2+) channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic ß-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca(2+)](i) or Ca(2+) efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca(2+)](i) induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca(2+) chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca(2+) signalling to ERK, we provide evidence that a sustained increase in Ca(2+) within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation.

Generation of epitope-tagged GPCRs.

The addition of one or more epitope tags to G-protein-coupled receptors (GPCRs) has facilitated a wide variety of studies on their structure and function. Epitope-tagging is achieved using relatively straightforward molecular techniques but requires careful consideration about the nature of the epitope tag and its location within the receptor. Here, we describe both the strategies and methodologies for the generation of epitope-tagged GPCRs. We highlight a range of possible techniques that depend upon the available starting material, the nature of the epitope to be incorporated, and suggest a strategy to ease the tagging of multiple receptor types.