Graeme F. Wilkinson

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor.

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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.

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, Ca2+ 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 Gαs 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 [Ca2+] 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.

Role of the signal peptide in the synthesis and processing of the glucagon-like peptide-1 receptor.

Background and purpose:  The glucagon‐like peptide‐1 receptor (GLP‐1R) belongs to Family B of the G protein‐coupled receptor superfamily and is a target for treatment of type 2 diabetes. Family B G protein‐coupled receptors contain a putative N‐terminal signal peptide, but its role in receptor synthesis and trafficking are unclear. Further, the signal peptide is not cleaved in at least one family member.

Ca2+ signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells

Human embryonic kidney (HEK)‐293 cells expressing recombinant Gαi‐coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca2+] ([Ca2+]i) in response to interleukin‐8 (IL‐8) following pre‐stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. Pre‐stimulation of cells with adenosine 5′‐triphosphate (ATP) revealed a substantial Ca2+ signalling component mediated by IL‐8 (Emax=83±8% of maximal ATP response, pEC50 of IL‐8 response=9.7±0.1). 1 μM 2‐methylthioadenosine 5′‐diphosphate (2MeSADP; P2Y1 selective) and 100 μM uridine 5′‐triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca2+]i elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca2+]i rapidly returned to basal levels. Both UTP and 2MeSADP increased the potency and magnitude of IL‐8‐mediated [Ca2+]i elevation but the effects of UTP (Emax of IL‐8 response increased to 50±1% of the maximal response to ATP, pEC50 increased to 9.8±0.1) were greater than those of 2MeSADP (Emax increased to 36±2%, pEC50 increased to 8.7±0.2). The potentiation of IL‐8‐mediated Ca2+ 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 Ca2+ signalling was apparent in the absence of extracellular Ca2+, demonstrating the release of Ca2+ from intracellular stores. Activation of P2Y1 and P2Y2 receptors also revealed Ca2+ signalling by an endogenously expressed, Gαs‐coupled β‐adrenoceptor. In conclusion, pre‐stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca2+ signalling by either recombinant CXCR2 or endogenous β‐adrenoceptors.

Full and Partial Agonists of Muscarinic M3 Receptors Reveal Single and Oscillatory Ca2+ Responses by β2-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 Gαq/11-coupled muscarinic M3 receptors facilitate Ca2+ signaling by Gαs-coupled β2-adrenoceptors. Measurement of changes in intracellular [Ca2+] demonstrated that noradrenaline released Ca2+ from thapsigargin-sensitive intracellular stores only during activation of muscarinic receptors. Agonists with low efficacy for muscarinic receptor-mediated Ca2+ responses facilitated cross-talk more effectively than full agonists. The cross-talk required Gαs and was dependent upon intracellular Ca2+ release channels, particularly inositol (1,4,5)-trisphosphate receptors. However, β2-adrenoceptor-mediated Ca2+ 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 Ca2+ signaling to noradrenaline. Thus, activation of muscarinic M3 receptors profoundly influences the magnitude and oscillatory behavior of intracellular Ca2+ signaling by β2-adrenoceptors. Although these receptor subtypes are often coexpressed and mediate contrasting acute physiological effects, altered oscillatory Ca2+ signaling suggests that cross-talk could influence longer term events through, for example, regulating gene transcription.

In Vitro Screening for Drug Repositioning

Drug repositioning or repurposing has received much coverage in the scientific literature in recent years and has been responsible for the generation of both new intellectual property and investigational new drug submissions. The literature indicates a significant trend toward the use of computational- or informatics-based methods for generating initial repositioning hypotheses, followed by focused assessment of biological activity in phenotypic assays. Another viable method for drug repositioning is in vitro screening of known drugs or drug-like molecules, initially in disease-relevant phenotypic assays, to identify and validate candidates for repositioning. This approach can use large compound libraries or can focus on subsets of known drugs or drug-like molecules. In this short review, we focus on ways to generate and validate repositioning candidates in disease-related in vitro and phenotypic assays, and we discuss specific examples of this approach as applied to a variety of disease areas. We propose that in vitro screens offer several advantages over biochemical or in vivo methods as a starting point for drug repositioning.

Regulation of the avidity of ternary complexes containing the human 5‐HT1A receptor by mutation of a receptor contact site on the interacting G protein α subunit

Fusion proteins were constructed between the human 5‐HT1A receptor and pertussis toxin‐resistant forms of both Gi1α and Go1α mutated at residue351 from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. Increasing concentrations of GDP inhibited binding of the agonist [3H]‐8‐OH‐DPAT but not the antagonist [3H]‐MPPF to each construct. The IC50 for GDP was greater for constructs containing isoleucine at residue351 of the G proteins compared to those with glycine at this position. The G protein antagonist suramin had similar effects to GDP on the binding of [3H]‐8‐OH‐DPAT. The proportion of 5‐HT1A receptor binding sites detected by [3H]‐MPPF that displayed high affinity for 8‐OH‐DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue351. The 5‐HT1A receptor displayed similar avidity of interaction with Gi1α and Go1α. These results indicate that a higher avidity ternary complex is formed between 8‐OH‐DPAT, the 5‐HT1A receptor and G proteins when isoleucine rather than glycine is located at residue351 of the interacting G protein.

Comparative Effects of the Endogenous Agonist GLP-1 7-36 Amide and a 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, Ca2+ 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 Gαs 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 [Ca2+] 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.

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, Ca2+ 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 Gαs 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 [Ca2+] 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.