Professor John Challiss

Tel:  0116 229 7146       Email:    jc36@le.ac.uk

Research Interests and Techniques

My lab focuses on signal transduction research with an emphasis on the use of molecular pharmacological approaches to understand how G protein-coupled receptors (GPCRs) shape short-term and longer-term responses to extracellular signals.  Current projects span interests in molecular neurobiology (using neurons and glial cells) and smooth muscle (vascular, airways and GU tract) physiology and pharmacology.  We are particularly interested in all aspects of muscarinic acetylcholine and metabotropic glutamate receptor physiology and pharmacology.

Figure 1

Confocal images of rat mesenteric arterial smooth muscle cells expressing an eGFP-PKCα construct and loaded with the Ca2+-sensitive fluorescent dye Fura-Red (Taken from Nelson et al. (2008) Am J Physiol – see Publications)

 

 

Background

A wide variety of G protein-coupled receptors (GPCRs) link to phospholipase C isoenzymes via  Gq/11 proteins to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and generate inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG).  The latter second messengers regulate intracellular Ca2+ concentration and protein kinase C activity, whereas PIP2 has emerged as an important regulator of processes including vesicle trafficking, cytoskeletal remodelling and ion channel modulation.  Research within the group has concentrated on signalling by by Gq/11-coupled GPCRs, including how Ca2+ (±PKC) can encode specific signals that regulate plastic changes in cellular responses and longer term decision-making in cells.  We have long-standing interests in muscarinic acetylcholine and metabotropic glutamate (particularly group 1 - mGlu1/mGlu5) receptors, as well as GPCRs for other mediators including lysophosphatidic acid, dopamine, endothelin-1, angiotensin II, histamine, oxytocin and neuromedin U.

A major advance in recent years has been the generation of 'biosensors' that allows signalling events to be visualized within single cells.  An example of such a biosensor is composed of the pleckstrin homology domain of PLCδ1 tagged with enhanced green fluorescent protein (eGFP-PH).  This construct can be introduced into cells by cDNA transfection and visualized within single cells by confocal fluorescence microscopy.  Under basal conditions eGFP-PH associates with PIP2 which is found predominantly in the inner leaflet of the plasma membrane.  On PLC activation the increase in IP3 concentration (and the decrease in PIP2 concentration) causes eGFP-PH to dissociate from the plasma membrane and accumulate in the cytoplasm.  This translocation can be tracked and quantified.  Using a variety of biosensors, as well as conventional Ca2+-sensitive fluorescent dyes (e.g. fluo-4), an array of signalling events can be visualized in living cells in real-time.

The signalling consequences of decreasing Gq/11α expression using short hairpin RNAi in HEK cell expressing the human M3 muscarinic acetylcholine receptor (Taken from Atkinson et al. (2006) Mol Pharmacol – see Publications)

The signalling consequences of decreasing Gq/11α expression using short hairpin RNAi in HEK cell expressing the human M3 muscarinic acetylcholine receptor (Taken from Atkinson et al. (2006) Mol Pharmacol – see Publications)

 

 

Current Research Projects

1.  Involvement of IP3 in encoding different patterns of change in [Ca2+]i.  We are investigating how different GPCRs are able to generate defined patterns of Ca2+ signal in cells.  These studies address (i) how different PLCs are recruited following GPCR activation, (ii) how feedback regulation at the level of the receptor-G protein-PLC, or at the level of the IP3R can regulate oscillatory Ca2+ patterning, (iii) how the Ca2+ signatures (together with other activated signalling pathways) can regulate longer term changes, for example through the regulation of transcription factors and transcriptional activity.  Some aspects of this work are already being investigated in neurons with a particular emphasis on how metabotropic and ionotropic inputs interact to regulate both IP3 and Ca2+ in the cell bodies and dendrites of hippocampal neurons.

2.  Regulation of GPCR activity by phosphorylation-dependent and -independent mechanisms.  Attenuation of signalling outputs following prolonged or recurrent receptor activation is referred to as desensitization.  Considerable evidence has accumulated to suggest that both G protein-coupled receptor kinases (GRKs) and second messenger-regulated kinases (e.g. PKC and PKA) can phosphorylate GPCRs.  We have studied this desensitization/internalization process for the M3 mACh receptor expressed endogenously in the SH-SY5Y neuroblastoma and have highlighted an important role for GRK6.  The use of the eGFP-PH biosensor for IP3 has also allowed us to study mACh receptor desensitization in single hippocampal neurons.  We are currently working on the relative roles that receptor phosphorylation and phosphorylation-independent mechanisms (for example, mediated by the ability of some GRKs to bind to and sequester Gαq/11 and/or Gβγ subunits) play in receptor regulation.

3. How does G protein subtype recruitment affect signalling patterns downstream of GPCRs?  We have previously presented evidence consistent with the group 1 mGlu receptors linking to Gi/o proteins as well as Gq/11 (and in the case of mGlu1 receptors Gs) proteins.  This 'promiscuous' coupling can result in a modified regulation of phosphoinositide turnover, and importantly in the case of the mGlu1 receptor a mechanism for linking to the extracellular signal-regulated protein kinase (ERK) MAPK pathway.  We are currently expanding these studies by using a number of approaches (e.g. RNAi) selectively to eliminate one or more Gα protein subtype and/or effector protein.

4. Pharmacological studies of constitutive activity and agonist-selective trafficking of signal at the mACh receptors.  There are at least three distinct objectives/projects in this area:  (i) Using mutant human mACh receptor subtypes we are investigating how increased constitutive activity affects a number of pharmacological properties, including inverse agonism, agonist-dependent and -independent G protein activation, receptor phosphorylation and downstream signalling, and the up-regulation of receptor expression by inverse agonists. (ii) Using either standard second messenger assays or a [35S]-GTPγS/immunoprecipitation approach, we are also investigating whether different mACh receptor agonists can activate different subsets of Gα protein subtypes. (iii) Some mACh receptor antagonists have been reported to display different affinity profiles in different cells/tissues leading to the idea that tissue selectivity may be possible between tissues that appear to express an identical GPCR subtype.  We have confirmed this phenomenon in guinea-pig tissues expressing M3 mACh receptors and are currently exploring possible mechanistic bases for such selectivity.

5. Explorations of GPCR 'cross-talk' in physiological regulation.  We are interested in how coincident activation of different GPCR subtypes can lead to inhibitory of facilitatory interactions at multiple levels in signal transduction pathways ('cross-talk').  At present this work focuses on cross-talk between Gq/11-coupled and Gi/o-coupled GPCRs (e.g. M2 and M3 mACh receptors), and Gq/11-coupled and Gs-coupled GPCRs (e.g. M3 mACh receptors and β2-adrenoceptors).

Research Expertise

1. Culturing, genetically manipulating and metabolically labelling mammalian cell-lines, and generating primary cultures e.g.  cerebellar granule cells, hippocampal neurones, astrocytes, smooth muscle cells derived from arterial (aortic/mesenteric), airway or uterine sources.

Immunocytochemical staining of rat cerebrocortical astrocytes grown in G5 supplement for 4-5 days (left panel, anti-mGlu5; middle panel, anti-GFAP, right panel, merge).

Immunocytochemical staining of rat cerebrocortical astrocytes grown in G5 supplement for 4-5 days (left panel, anti-mGlu5; middle panel, anti-GFAP, right panel, merge).

2. A wide variety of molecular pharmacological techniques including: radioligand binding; [35S]-GTPγS binding assays and Gα-specific immunoprecipitation/immunocapture; second messenger assays (cAMP, cGMP, IP3) and all aspects of assessing phosphoinositide turnover (including [3H]-PI/PIP/PIP2 separation, etc.). PLC/PLA2/PLD assays. Cell growth and proliferation assays (e.g. [3H]-thymidine incorporation). Standard SDS-PAGE/western blot approaches, including experience with a wide array of signalling component antibodies (for receptors, G proteins, effectors, protein kinases and transcription factors). Measurement of ERK/JNK/p38 through phospho-specific antibodies and [32P]-ATP/kinase activity assays. Immunocytochemistry.

3. Population and single cell imaging approaches: confocal fluorescence microscopy to assess Ca2+ and biosensor reporters for second messengers/signaling intermediates using translocating probes (e.g. eGFP-PH) and FRET biosensors.

4. Molecular manipulation of protein activity/function in intact cells through the use of dominant-negative protein expression, antisense and RNAi/siRNA approaches.

Research Group and Funding

Present Group Members

Dr Elena Christofidou
Mr Rajendra Mistry
Mr Xianguo Jiang
Dr Paul Glynn

Ex Lab Alumni

Post-doctoral

John Mackrill, Helen Sherriffs, Jonathon Willets, Mark Nash, Kenneth Young, Daniela Billups, Danijela Markovic, Hasib Salah-Uddin, Gavin Morris, Melissa Jordan, Jennifer Brignell, Carl Nelson, Sophie Bradley, Edith Gomez, Craig Nash

Doctoral/Masters

Emma Whitham (PhD, 1991), Anthony Morgan (PhD, 1992), Stephen Jenkinson (PhD, 1993), David Adams (PhD, 1994), Peter Simpson (PhD, 1995), Nageen Hashmi (MRes, 1996), Alan Carruthers (PhD, 1997), Donna Boxall (PhD, 1998), Rick Davis (PhD, 1999), Elizabeth Akam (PhD, 1999), Kirsti Hill (MRes, 1999), Paul Wylie (PhD, 2000), Ruth Saunders (PhD, 2000), David Hornigold (PhD, 2001), Jules Selkirk (PhD, 2001), Drew Burdon (PhD, 2002), Sukhwinder Thandi (PhD, 2004), Carl Nelson (PhD, 2004), Mark Dowling, (PhD, 2004), Helen Warwick (PhD, 2005), Peter Atkinson (PhD, 2005), Paula Bartlett (PhD, 2006), Elizabeth Rosethorne (PhD, 2007), David Pier (PhD, 2007); Rachel Thomas (PhD, 2009); Sophie Bradley (PhD, 2011); George Shehatou (PhD, 2011); Aminah Loonat (MRes, 2011); Khaled Al-Hosaini (PhD, 2011); David Sweeney(PhD, 2011), Ka Ming Law (MRes, 2012); Rupert Satchell (PhD, 2012); Manish Asiani (MRes, 2013); Marie Valente (PhD, 2014); Leonarda di Candia (PhD, 2015)

Current Funding

British Heart Foundation (Project Grant)

 

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Contact Details

Department of Molecular and Cell Biology
Henry Wellcome Building
Lancaster Road
Leicester
LE1 7RH (Postal)

LE1 7HB (Sat Nav/Online maps)

T:  +44(0)116 229 7038
F:  +44(0)116 229 7123
MolCellBiol@le.ac.uk

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Redfearn Lecture 2017

To Be Confirmed