Furness Laboratory

Chemical biology of GPCR signal transduction

Project areas

Research focus


Fluorescent confocal microscopy showing various kidney cell structures with a GPCR at the cell surface in red.

The membrane at the surface of  cells is their defining feature, maintaining internal organisation in the face  of the disorder of the environment. All cellular requirements and communication  must cross this membrane. G protein-coupled receptors (GPCRs) represent the  largest  class  of molecular machines that provide the means of communication  across this barrier. This family of receptors allow cells to sense local and  distant signals sent from other cells, nutrients in the gastro-intestinal  tract, odours & tastes and even allow us to see.  Chemical  biology  is a holistic approach to understanding the underlying molecular basis of  biological events.

My research seeks to develop a  deeper understanding of the molecular basis for communication across the cell  membrane by GPCRs using a variety of biochemical, molecular, cell biological  and pharmacological methods – in essence a chemical biology approach. This  molecular  description  of how GPCRs work is used to inform our understanding of  whole organism physiology and pathology which we pursue via active  collaborations.

How are different signals sensed by single GPCRs


TIRF (total internal reflection fluorescence microscopy) tracking of the dynamic movements of single GPCRs. This movie shows real time movement of a GPCR at the cell surface.

Some GPCRs are able to sense more than one kind of signal. In these cases the cell, via the GPCR, must be able to distinguish these signals. Very good pharmacological models have been developed that provide an explanation of how different signals act at a single GPCR and are differentiated within the cell. This is commonly known as biased signalling. The current pharmacological understanding of this phenomenon assumes that the shape adopted by the GPCR differs depending on which signal is present and that somehow this different shape is communicated to within the cell. There is, however, no molecular description of this process. We have been using a model GPCR and 2 slightly different chemicals that are sensed by it to probe the underlying molecular basis for this phenomenon. We have applied novel techniques borrowed from biologists who study cellular respiration, intracellular sensors to monitor the cellular response in real time as well as tracking single molecules of the receptor and its chemical signal using super-resolution microscopy. Understanding the underlying molecular basis of biased signalling will provide insights that can be applied to developing better drugs and a short video describing some of this work can be found here.

The atomic level detail of GPCR structure


Atomic resolution structure of a GPCR ternary complex. In green is the GPCR and in yellow, red and teal are the components of the signaling machine.

GPCRs are highly complex molecular  machines. For a signal to be transmitted into the cell the GPCR must adopt a  shape that performs two simultaneous roles. Firstly, the shape must  specifically recognise the signal coming from the environment. Secondly the  shape  must  induce a change in the shape of a signalling machine inside the cell  in order to effect the switching of this machine from one state to another  (e.g. off state to on state).

The complex which contains the activating signal,  the GPCR and the intracellular signalling machine is known as the ternary  complex. This ternary complex may exist for only fractions of a second in  physiological situations. While many of the projects in my lab involve  the  understanding  of dynamic changes in GPCRs this project involves trying to  understand the atomic detail of the shape of a GPCR in its ternary complex.

To capture this tiny moment in time  in the dynamic life of a GPCR we must make large amounts of the various  components of the ternary complex and bring them together under special  conditions that promote the formation of crystals that freeze this moment and  allow  us  to capture it via x-ray crystallography. This is a large undertaking  and collaboration and it will lead to a better understanding of the way in  which GPCRs work.

How GPCRs change their shape in response to signals


A cartoon depicting the changes in  shape that a GPCR undergoes in order to transmit signals across the cell  membrane.

For signals to traverse the cell  membrane there must be a change in shape of the sensing GPCR that is  transmitted to the inside of the cell. For us to be able to understand the  basis of this phenomenon we are investigating the molecular details of the different  shapes  that  GPCRs can adopt and, importantly, how they are able to transition  between these different shapes.  Using a  model GPCR we are inserting environmentally sensitive probes whose fluorescence  changes depending on the neighbouring environment. This allows us  to  track the  different shapes that this GPCR adopts according to what signal it is sensing  and how that signal is then transmitted.

We also have an on-going program  developing  antibodies to model GPCRs  that distinguish between the different shapes  adopted by a single GPCR. As an offshoot of this program we have identified an  antibody that specifically identifies cells responsible  for  initiating a type  of brain tumour and are actively pursuing this as a potential method to treat  this deadly disease (see below). The understanding of the different shapes that  GPCRs adopt and how they transition between these shapes will provide molecular  level  information  that can be used to better understand various pathological  states as well as design drugs with better specificity and lower side effects.

The role and targeting of the Calcitonin receptor to treat brain tumours


A confocal image of a tumour biopsy from a patient with Glioblastoma. In red are the malignant glioma cells, in pink are endothelial cells and in green are pericyte precursors.

The Calcitonin receptor is a GPCR  that is involved in bone physiology. In the course if developing antibodies  directed against the Calcitonin receptor (see above) Dr.  Peter Wookey  identified  the presence of high levels of Calcitonin receptor on  cells  that initiate a  type of brain tumour known as Glioblastoma.  This type of brain tumour has particularly poor prognosis with usual survival  from diagnosis of less than 2 years.

As part of a large international  collaboration I have been developing a toxin that uses a Calcitonin receptor  directed antibody and is highly specific against the cells of the brain tumour.  We have shown that this toxin is highly effective at killing brain tumour cells  in  culture  and are currently developing experiments to show that this is also  true in animal models of this tumour. Any improved therapy for this deadly type  of tumour will be a major advance for patients.

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Key publications

Research  papers

Liang YL, Khoshouei M, Radjainia M, Zhang Y, Glukhova A, Tarrasch J, Thal DM, Furness SGB, Christopoulos G, Coudrat T, Danev R, Baumeister W, Miller LJ, Christopoulos A, Kobilka BK, Wootten D, Skiniotis G, Sexton PM. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature. 2017 Apr 24. doi: 10.1038/nature22327. PubMed PMID: 28437792.

Furness SG, Liang YL, Nowell CJ, Halls ML, Wookey PJ, Dal Maso E, Inoue A, Christopoulos A, Wootten D, Sexton PM. Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy. Cell. 2016 Oct 20;167(3):739-749.e11. doi: 10.1016/j.cell.2016.09.021. PubMed PMID: 27720449.

Furness SG, Hare DL, Kourakis A, Turnley AM, Wookey PJ. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death. Cell Death Discov. 2016 Oct 10;2:16062. PubMed PMID: 27777788; PubMed Central PMCID: PMC5056446.

Wootten D, Reynolds CA, Smith KJ, Mobarec JC, Koole C, Savage EE, Pabreja K, Simms J, Sridhar R, Furness SG, Liu M, Thompson PE, Miller LJ, Christopoulos A, Sexton PM. The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism. Cell. 2016 Jun 16;165(7):1632-43. doi:10.1016/j.cell.2016.05.023. PubMed PMID: 27315480; PubMed Central PMCID: PMC4912689.

Cook AE, Mistry SN, Gregory KJ, Furness SG, Sexton PM, Scammells PJ, Conigrave AD, Christopoulos A, Leach K. Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics. Br J Pharmacol. 2015 Jan;172(1):185-200. doi: 10.1111/bph.12937. PubMed PMID: 25220431; PubMed Central PMCID: PMC4280977.

Andreassen KV, Hjuler ST, Furness SG, Sexton PM, Christopoulos A, Nosjean O, Karsdal MA, Henriksen K. Prolonged calcitonin receptor signaling by salmon, but not human calcitonin, reveals ligand bias. PLoS ONE. 2014 Mar 18;9(3):e92042. doi: 10.1371/journal.pone.0092042

Savage EE, Wootten D, Christopoulos A, Sexton PM, Furness SG. A simple method to generate stable cell lines for the analysis of transient protein-protein interactions. Biotechniques. 2013 Apr;54(4):217-21. doi: 10.2144/000114013. PubMed PMID: 23581469.

Harikumar KG, Wootten D, Pinon DI, Koole C, Ball AM, Furness SG, Graham B, Dong M, Christopoulos A, Miller LJ, Sexton PM. Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery. Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):18607-12. doi: 10.1073/pnas.1205227109. Epub 2012 Oct 22. PubMed PMID: 23091034; PubMed Central PMCID: PMC3494884.

Willard FS, Wootten D, Showalter AD, Savage EE, Ficorilli J, Farb TB, Bokvist K, Alsina-Fernandez J, Furness SG, Christopoulos A, Sexton PM, Sloop KW. Small molecule allosteric modulation of the glucagon-like Peptide-1 receptor enhances the insulinotropic effect of oxyntomodulin. Mol Pharmacol. 2012 Dec;82(6):1066-73. doi: 10.1124/mol.112.080432. Epub 2012 Aug 28. PubMed PMID: 22930710.

Wookey PJ, McLean CA, Hwang P, Furness SG, Nguyen S, Kourakis A, Hare DL, Rosenfeld JV. The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172. Histopathology. 2012 May;60(6):895-910. doi: 10.1111/j.1365-2559.2011.04146.x. Epub 2012 Feb 15. PubMed PMID: 22335784.

Reviews

Furness SG, Wootten D, Sexton PM. What determines the magnitude of cellular response for activation of G protein-coupled receptors? Cell Cycle. 2017 Jan 5:0. doi: 10.1080/15384101.2016.1271634. [Epub ahead of print] PubMed PMID: 28055294.

Pabreja K, Mohd MA, Koole C, Wootten D, Furness SG. Molecular mechanisms underlying physiological and receptor pleiotropic effects mediated by GLP-1R activation. Br J Pharmacol. 2014 Mar;171(5):1114-28. doi: 10.1111/bph.12313. PubMed PMID: 23889512.

Koole C, Pabreja K, Savage EE, Wootten D, Furness SG, Miller LJ, Christopoulos A, Sexton PM. Recent advances in understanding GLP-1R (glucagon-like peptide-1 receptor) function. Biochem Soc Trans. 2013 Feb 1;41(1):172-9. doi: 10.1042/BST20120236. Review. PubMed PMID: 23356279.

Furness SG, Wootten D, Christopoulos A, Sexton PM. Consequences of splice variation on Secretin family G protein-coupled receptor function. Br J Pharmacol. 2012 May;166(1):98-109. doi: 10.1111/j.1476-5381.2011.01571.x. Review. PubMed PMID: 21718310; PubMed Central PMCID: PMC3415641.

Furness SG & Whelan F. The pleiotropy of dioxin toxicity—xenobiotic misappropriation of the aryl hydrocarbon receptor's alternative physiological roles. Pharmacol Ther. 2009 Dec;124(3):336-53. doi: 10.1016/j.pharmthera.2009.09.004. Epub 2009 Sep 23. PubMed PMID: 19781569.

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Collaborations

How are different signals sensed by single GPCRs

Dr Denise Wootten, Drug Discovery Biology, MIPS

Dr Michelle Halls, Drug Discovery Biology, MIPS

Prof Auka Inoue, Tohoku University, Japan

Dr Peter Wookey, University of Melbourne, Austin Health

The atomic level detail of GPCR structure

Prof Patrick Sexton, Drug Discovery Biology, MIPS

Dr Denise Wootten, Drug Discovery Biology, MIPS

Prof Brian Kobilka, Stanford University, U.S.A.

Prof GeorgiosSkiniotis, University of Michigan, U.S.A.

How GPCRs change their shape in response to signals

Prof Patrick Sexton, Drug Discovery Biology, MIPS

Dr Denise Wootten, Drug Discovery Biology, MIPS

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Lab members

Ms Anna Ostrovskaya
PhD student

Ms Emma Dal Maso
PhD student

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Funding

  • NHMRC  Project Grant (2013 – 2015) Understanding  the structural basis for Family B G protein-coupled receptor function

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