Dr Gary Willars
|Tel: 0116 229 7147 Email: email@example.com|
Research Interests and Techniques
The overall aim of our work is to increase understanding of the activation and regulation of intracellular signalling mediated by G-protein-coupled receptors (GPCRs). Previous work has focused on the mechanisms involved in the feedback regulation of signalling, particularly in relation to those receptors that activate phospholipase C. This has included consideration of such aspects as: Ca2+ regulation of phospholipase C; protein kinase C-dependent feedback; receptor phosphorylation; regulation of Ins(1,4,5)P3 receptor expression; the regulation of phosphoinositide supply; the role of regulators of G-protein signalling (RGS) proteins in signal attenuation. Current projects within the laboratory focus on:
1) Glucagon-like peptide 1 (GLP-1) receptor signalling
Processing of progucagon within L cells of the intestine forms a number of peptides including GLP-1. This is secreted following nutrient ingestion and acts as a major incretin hormone, substantially enhancing the postprandial insulin response by enhancing glucose-dependent insulin release from pancreatic β-cells. GLP-1 mediates its effects via a Gαs–coupled, Family B GPCR.
Given the enhanced glucose-dependent insulin release, the GLP-1R is an attractive target for the treatment of type 2 diabetes mellitus, particularly as the risk of drug-induced hypoglycaemia is less than with current therapies. Furthermore, GLP-1 exerts additional pancreatic and extra-pancreatic anti-diabetogenic effects that may enhance its clinical efficacy. Unfortunately from a therapeutic perspective, GLP-1 is rapidly degraded by dipeptidyl peptidase IV and there has, therefore, been a focus on the development of more stable analogues of GLP-1 and small molecule ligands.
Our recent work using site-directed mutagenesis has examined key residues involved in ligand binding or receptor activation, particularly within the transmembrane domains of the receptor. This work has contributed to the generation of a model of the 3-D structure of the GLP-1-bound GLP-1R. This work is continuing but now considering aspects of receptor structure that may contribute to biased agonism, orthosteric and allosteric receptor activation and interactions between orthosteric and allosteric sites.
Other work has examined both orthosteric and allosteric activation of the GLP-1R their impact on aspects such as receptor internalisation and trafficking and interactions between ligands at these different sites. Of particular interest are the recent observations that combinations of a small molecule ago-allosteric compound and orthosteric antagonists or weak partial agonists can markedly synergise. Given that degradation products of GLP-1 are antagonists or weak partial agonists but often present at much higher concentrations than the active peptide, such interactions could provide a novel therapeutic approach.
GLP-1 binds with high affinity to the GLP-1R and as a consequence of receptor phosphorylation and subsequent arrestin binding, internalises into the endosomal compartment. The current paradigm suggests that such internalisation of the receptor is critical for dephosphorylation, receptor recycling and resensitisation. However, for many receptors with peptide ligands such as the GLP-1R, the ligand binds with high affinity and co-internalises with the receptor.
The current scheme of receptor processing following internalisation does not really consider any role for the ligand or ligand processing other than the suggesting that the acidic conditions of the endosome may enhance ligand-dissociation. Recent evidence has emerged that the processing of peptide, by for example endosmally-located endothelin converting enzxme-1 (ECE-1), may be required to facilitate receptor recycling. Aspects of our current work are focusing on the potential role of ECE-1 in the processing of internalised GLP-1 nd the consequences of this on resensitisation of the signalling pathways.
2) Pro-insulin C-peptide-mediated signalling and function
The diabetes theme is continued in collaborative work with Professor Nigel Brunskill (Department of Infection, Immunity and inflammation) and Dr Steve Ennion (Department of Cell Physiology and Pharmacology) in which the potential cellular signalling and roles of pro-insulin C-peptide are being investigated, particularly in relation to the nephropathic changes associated with long-standing, poorly controlled diabetes. We are particularly keen to identify a receptor that mediates the effects of C-peptide and have devised a number of strategies to address this issue.
3) Neuromedin U receptors
Neuromedin U (NmU) belongs to a family of peptides termed neuromedins originally isolated in the mid 1980s. NmU is highly conserved across species, with mammalian versions ranging from 8 to 25 amino acids and all having identical C-terminal heptapeptides. Although the pathophysiological functions remain to be precisely defined, NmU may have important roles in the regulation of: smooth-muscle contraction; blood pressure and regional blood flow; the stress response and; feeding and energy expenditure. NmU mediates these diverse effects through two distinct family A GPCRs, NMU1 and NMU2, that show differential distribution. More recently, the neuropeptide neuromedin S (NmS) has also been identified in a number of species as an endogeneous ligand of these receptors, being expressed centrally and binding to NMU1 and NMU2 with similar affinity to NmU. NmS peptides are found in many species and are generally of similar length (33-36 amino acids), with the C-terminal heptapeptide of NmS being identical to that of mammalian forms of NmU. The precise roles of these different ligands and whether any signalling differences exist remains to be defined.
We have extensively characterised aspects of the signalling by recombinant and endogenously expressed NMU and examined contractile effects on smooth muscle. As part of this work we have shown that the peptide ligands of NMU bind essentially irreversibly and internalise along with the receptor. Our current work is assessing the consequence of such internalisation on aspects of receptor signalling and regulation and considering potential differences between NmU and NmS. As with the GLP-1R, we are also examining the impact of ECE-1 activity on receptor signalling and particularly receptor resensitisation.
- Determination of GPCR- mediated signalling:
- Measurement of [Ca2+]i in populations of both adherent and suspended cells
- Single cell imaging of [Ca2+]i using standard imaging techniques and confocal microscopy
- 45Ca2+ release from intracellular stores of permeabilised cells
- Determination of phospholipase C activity by either the accumulation of [3H]-inositol phosphates in lithium-blocked cells or Ins(1,4,5)P3 mass measurement
- Single cell imaging of Ins(1,4,5)P3 and diacylglycerol formation using biosensors
- Measurement of phosphoinositides
- Measurement of cAMP
- Determination of receptor expression by radioligand binding.
- Determination of protein expression by Western blotting.
- Determination of in vivo protein phosphorylation.
- Standard molecular biology techniques, including mutagenesis.
- Use of fluorescent ligands to assess receptor binding and internalisation.
- Cellular localisation and real-time movement of GFP-tagged proteins.
- Development and maintenance of clonal cell lines and preparation of a number of primary cultures.
Research Group and Funding
Present group members
Dr Naichang Li (Visiting worker from Tianjin Medical University, China)
Varadarajan, S., Tanaka, K., Smalley, J.L., Bampton, E.T.W., Pellecchia, M., Dinsdale, D., Willars, G.B. and Cohen, G.M. (2013) Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis. PLoS ONE 8(2): e56603. doi:10.1371/journal.pone.0056603
Tovey, S.C., Brighton, P.J., Bampton, E.T.W., Huang, Y. and Willars, G.B. (2013) Confocal microscopy: theory and applications for cellular signaling In: Methods in Molecular Biology Vol. 937: Calcium Signaling Protocols 3rd Ed. (Lambert D.G. and Rainbow R. eds.) Humana Press Inc., Totowa, NJ. pp. 51-93.
Li, N., Lu, J. and Willars, G.B. (2012) Allosteric modulation of the activity of the glucagon-like peptide-1 (GLP-1) metabolite GLP-1 9-36 amide at the GLP-1 receptor. PLoS ONE 7 e47936 1-10
Selway, J., Rigatti, R., Storey, N., Lu, J., Willars, G.B. and Herbert, T.P. (2011) 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. PLoS ONE 7 e330004 1-10
Coopman, K., Wallis, R., Robb, G., Brown, A., Wilkinson, G.F., Timms, D. and Willars, G.B. (2011) Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor. Molecular Endocrinology 25, 1804-1818.
Xie, J., Ponuwei, G.A., Moore, C.E., Willars, G.B., Tee, A.R. and Herbert T.P. (2011) cAMP inhibits mammalian target of rapamycin complex-1 and -2 (mTORC1 and 2) by promoting complex dissociation and inhibiting mTOR kinase activity. Cellular Signalling 23, 1927-1935
Moore, C.E., Omikorede, O., Gomez, E., Willars, G.B. and Herbert, T.P. (2011)
PERK Activation at low glucose concentration is mediated by SERCA pump inhibition and confers preemptive cytoprotection to pancreatic ß-cells. Molecular Endocrinology 25, 315-326.
Huang, Y. and Willars, G.B. (2011) Generation of epitope-tagged GPCRs. In: Methods in Molecular Biology Vol 746: GPCR Signal Transduction Protocols 3rd Edition, (Willars, G.B. and Challiss, R.A.J. eds.) Humana Press Inc., Totowa, NJ. pp 53-84.
Coopman, K., Huang, Y., Johnston, N., Bradley, S.J., Wilkinson, G.F. and Willars, G.B. (2010) 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. Journal of Pharmacology and Experimental Therapeutics 334, 795-808.
Day, C.E., Guillen, C., Willars, G.B. and Wardlaw, A.J. (2010) Characterization of the migration of lung and blood T cells in response CXCL12 in a three-dimensional matrix. Immunology 130, 564-571.
Hills, C.E., Willars, G.B. and Brunskill, N.J. (2010) Pro-insulin C-peptide antagonizes the pro-fibrotic effects of TGF- ß1 via upregulation of retinoic acid and HGF related signaling pathways. Molecular Endocrinology 24, 822-831.
Huang, Y., Wilkinson, G.F. and Willars, G.B. (2010) Role of the signal peptide in the synthesis and processing of the glucagon-like peptide-1 receptor. British Journal of Pharmacology 159, 237-251.
Kurian, N., Hall, C.J., Wilkinson, G.F., Sullivan, M., Tobin, A.B. and Willars, G.B. (2009) Full and partial agonists of muscarinic M3 receptors reveal single and oscillatory Ca2+ responses by β2-adrenoceptors. Journal of Pharmacology and Experimental Therapeutics 330, 502-512.
Hills, C.E., Al-Rasheed, N., Al-Rasheed, N., Willars, G.B. and Brunskill N.J. (2009) C-Peptide reverses TGF-1-Induced changes in renal proximal tubular cells: implications for the treatment of diabetic nephropathy. American Journal of Physiology: Renal Physiology 296, F614-F621.
Mayer SI, Willars GB, Nishida E, Thiel G. (2008) Elk-1, CREB, and MKP-1 regulate Egr-1 expression in gonadotropin-releasing hormone stimulated gonadotrophs. J. Cell. Biochem. 105, 1267-1278.
Hayabuchi Y, Willars GB, Standen NB, Davies NW (2008) Insulin-like growth factor-I inhibits rat arterial KATP channels through pI 3-kinase. Biochem. Biophys. Res. Comm. 374, 742-746.
Brighton PJ, Wise A, Dass NB, Willars GB. (2008) Paradoxical behaviour of neuromedin U in isolated smooth muscle cells and intact tissue. J. Pharmacol. Exp. Ther. 325, 154-164.
Karakoula A, Tovey SC, Brighton PJ, Willars GB (2008) Lack of receptor-selective effects of either RGS2, RGS3 or RGS4 on muscarinic M3- and gonadotropin-releasing hormone receptor-mediated signalling through Gαq/11. Eur. J. Pharmacol. 587, 16-24.
Cockerill SL, Tobin AB, Torrecilla I, Willars GB, Standen NB and Mitcheson JS. (2007) Modulation of hERG potassium currents in HEK-293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits. J. Physiol. 581, 479-493.
Willars GB. (2006) Mammalian RGS proteins: multifunctional regulators of cellular signalling. Sem. Cell Develop. Biol. 17, 363-376.
Al-Rasheed NM, Willars GB and Brunskill NJ. (2006) C-peptide signals via Gαi to protect against TNF-α-mediated apoptosis of opossum kidney proximal tubular cells J. Am. Soc. Nephrol. 17, 986-995.
Tovey SC, Brighton PJ and Willars GB. (2005) Confocal microscopy: theory and applications for cellular signaling In: Methods in Molecular Biology vol. 312: Calcium Signaling Protocols 2nd Ed. (Lambert DG ed.) Humana Press Inc., Totowa, NJ. pp. 57-85.
Tovey SC and Willars GB. (2004) Single-cell imaging of intracellular Ca2+ and phospholipase C activity reveals that RGS 2, 3 and 4 differentially regulate signaling via the Gαq/11-linked muscarinic M3 receptor. Mol. Pharmacol.66 1453-1464.
Brighton PJ, Szekeres PG, Wise A and Willars GB. (2004) Signaling and ligand binding by recombinant neuromedin U receptors: evidence for dual coupling to Gαq/11 and Gαi and an irreversible ligand-receptor interaction. Mol. Pharmacol. 66 1544-1556.
Al-Rasheed NM, Chana RS, Baines RJ, Willars GB and Brunskill NJ. (2004) Ligand independent activation of peroxisome proliferator activated receptor-γ by insulin and C-peptide in kidney proximal tubular cells: dependent on phosphatidylinositol 3-kinase activity. J. Biol. Chem. 279 49747-49754.
Al-Rasheed NM, Meakin F, Royal EL, Lewington AJ, Brown J, Willars GB and Brunskill NJ. (2004) Potent activation of multiple signalling pathways by C-peptide in kidney proximal tubular cells. Diabetologia 47, 987-997.
Receptor Signal Transduction Protocols. (2004) Methods in Molecular Biology Series. Humana Press. Vol. 259. Eds. Willars GB and Challiss RAJ. (Book) ISBN 1-58829-329-7
Brighton P, Szekeres PG and Willars GB. (2004) Neuromedin U and its receptors: structure, function and physiological roles. Pharmacol. Rev. 56, 231-248.
Werry TD, Wilkinson GF and Willars GB. (2003) Mechanisms of crosstalk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+ Biochem. J. 374, 281-296.
Werry TD, Wilkinson GF and Willars GB. (2003) Crosstalk between P2Y2 nucleotide receptors and CXCR2 resulting in enhanced Ca2+ signalling involves enhancement of phospholipase C activity, and is enabled by incremental Ca2+ release in HEK cells. J. Pharmacol. Exp. Ther. 307, 661-669.
Witherow DS, Tovey SC, Willars GB and Slepak VZ. (2003) G b 5•RGS7 inhibits G a q -mediated signalling via a direct protein-protein interaction. J. Biol. Chem. 278, 21307-31313.