Receptor Transducer Coupling

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 affect 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 of 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

Ostrovskaya A, Hick C, Hutchinson DS, Stringer BW, Wookey PJ, Wootten D, Sexton PM, Furness SGB. Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas. BMC Cancer. 2019 Feb 18;19(1):157. doi: 10.1186/s12885-019-5369-y.

Dal Maso E, Glukhova A, Zhu Y, Garcia-Nafria J, Tate CG, Atanasio S,Reynolds CA, Ramírez-Aportela E, Carazo J-M, Hick CA, Furness SGB, Hay DL, Liang Y-L, Miller LJ, Christopoulos A, Wang M-W, Wootten D, and Sexton PM. The Molecular Control of Calcitonin Receptor Signaling. ACS Pharmacology & Translational Science. 2019;:acsptsci.8b00056.

Furness SGB, Christopoulos A, Sexton PM, Wootten D. Differential engagement of polar networks in the glucagon-like peptide 1 receptor by endogenous variants of the glucagon-like peptide 1. Biochem Pharmacol. 2018 Aug 25;156:223-240. doi: 10.1016/j.bcp.2018.08.033.

Liang Y-L, Zhao P, Draper-Joyce CJ, Baltos J-A, Glukhova A, Truong TT, May LT, Christopoulos A, Wootten D, Sexton PM, and Furness SGB. Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination. ACS Pharmacology & Translational Science 2018 1(1), 12-20

Draper-Joyce CJ, Khoshouei M, Thal DM, Liang YL, Nguyen ATN, Furness SGB, Venugopal H, Baltos JA, Plitzko JM, Danev R, Baumeister W, May LT, Wootten D, Sexton PM, Glukhova A, Christopoulos A. Structure of the adenosine-bound human adenosine A(1) receptor-G(i) complex. Nature. 2018 Jun;558(7711):559-563. doi: 10.1038/s41586-018-0236-6. PubMed PMID: 29925945.

Liang YL, Khoshouei M, Glukhova A, Furness SGB, Zhao P, Clydesdale L, Koole C, Truong TT, Thal DM, Lei S, Radjainia M, Danev R, Baumeister W, Wang MW, Miller LJ, Christopoulos A, Sexton PM, Wootten D. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature. 2018 Mar 1;555(7694):121-125. doi: 10.1038/nature25773. PubMed PMID: 29466332.

Dal Maso E, Zhu Y, Pham V, Reynolds CA, Deganutti G, Hick CA, Yang D, Christopoulos A, Hay DL, Wang MW, Sexton PM, Furness SGB*, Wootten D*. Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy. Biochem Pharmacol. 2018 Apr;150:214-244. doi: 10.1016/j.bcp.2018.02.005. PubMed PMID: 29454620; PubMed Central PMCID: PMC5908784.

Dal Maso E, Just R, Hick C, Christopoulos A, Sexton PM, Wootten D, Furness SGB. Characterization of signalling and regulation of common calcitonin receptor splice variants and polymorphisms. Biochem Pharmacol. 2018 Feb;148:111-129. doi: 10.1016/j.bcp.2017.12.016. PubMed PMID: 29277692.

Gilabert-Oriol R*, Furness SGB*, Stringer BW, Weng A, Fuchs H, Day BW, Kourakis A, Boyd AW, Hare DL, Thakur M, Johns TG, Wookey PJ (2017). Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma. Cancer Immunol Immunother. 2017 Sep;66(9):1217-1228. doi: 10.1007/s00262-017-2013-z. PubMed PMID: 28501939.

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 Jun 1;546(7656):118-123. 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. 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. 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. PubMed PMID: 22335784.

Reviews

Furness SG, Sexton PM (2017). Coding GPCR-G protein specificity. Cell Res. 2017 Oct;27(10):1193-1194. doi: 10.1038/cr.2017.92. PubMed PMID: 28695889.

Furness SG, Wootten D, Sexton PM. What determines the magnitude of cellular response for activation of G protein-coupled receptors? Cell Cycle. 2017 Mar 4;16(5):392-394. doi: 10.1080/15384101.2016.1271634. 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. PubMed PMID: 19781569.

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Collaborations

How are different signals sensed by single GPCRs and their transducers

A/Prof Denise Wootten, Drug Discovery Biology, MIPS

Prof Scott Prosser, University of Toronto, Canada

Prof Paul Gooley, Bio21 Institute, Melbourne

Dr Chris Ritchie, School of Chemistry, Monash, Clayton

The atomic level detail of GPCR structure

Prof Patrick Sexton, Drug Discovery Biology, MIPS

A/Prof Denise Wootten, Drug Discovery Biology, MIPS

Dr Lynn Liang, Drug Discovery Biology, MIPS

How GPCRs change their shape in response to signals

Prof Patrick Sexton, Drug Discovery Biology, MIPS

A/Prof Denise Wootten, Drug Discovery Biology, MIPS

Targeting the calcitonin receptor in brain cancer

Dr Peter Wookey, University of Melbourne, Austin Health

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Lab members

Ms Anna Ostrovskaya
PhD student

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Funding

  • ARC Future Fellowship (2019-2022) The molecular basis for efficacy at G protein-coupled receptors (FT180100543)
  • NHMRC Project Grant (2017-2021) A structural understanding of class B G protein-coupled receptor function (APP1120919)
  • NHMRC Project Grant (2013 – 2015) Understanding the structural basis for Family B G protein-coupled receptor function (APP1059015)

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