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 proglucagon 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.

Current projects are examining various aspects of the structure-function relationships between ligand and receptor; the signalling pathways regulated by receptor activation; receptor synthesis, trafficking and internalisation; the use of synthetic small molecule ligands as receptor agonists. 

The diabetes theme is continued in collaborative work with Professor Nigel Brunskill (Department of Infection, Immunity and inflammation) 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.

2) Signalling by 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.

We have characterised aspects of the signalling by recombinant and endogenously expressed receptors and examined contractile effects on smooth muscle. We have shown that NmU binds essentially irreversibly to recombinantly expressed receptors, resulting in a single round of signalling but that in smooth muscle, repetitive signalling is possible. Current work is examining this paradox and further seeking to characterise the regulation (eg. desensitisation, signal termination) of these receptors.

3) Receptor crosstalk

Cells are rarely exposed to a single type of receptor agonist. Crosstalk between different receptors and their signalling pathways provides mechanisms for integrating multiple signalling events and allowing appropriate regulation of cellular responses. Such crosstalk often results in heterologous desensitisation but can also either potentiate or facilitate signalling by alternative pathways. One example of such positive crosstalk is that in which only in the presence of Gaq/11 activation can the activation of Gai- or Gas-coupled GPCRs elevate the intracellular [Ca2+]  ([Ca2+]i).

We have explored this crosstalk extensively using both endogenously- and recombinantly-expressed receptors. Current work is focussing on examining the potential consequences of altered Ca2+ signalling and exploring potential pathophysiological consequences.

Techniques

• 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.
• Immunocytochemistry.
• 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

Khaled Al-Hosaini
Mr Neil Johnston  
Jing Lu

Recent Publications 

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.