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Cell Surface Receptors (G protein-coupled receptors)

Node(s): The Walter and Eliza Hall Institute University of Wollongong Monash University

Over 800 genes encoding GPCRs are present in the human genome, with splice variants, differential post-translational processing and formation of heteromeric complexes between distinct GPCRs, as well as between GPCRs and other modulatory proteins (e.g. receptor activity-modifying proteins; RAMPs), greatly amplifying the repertoire of GPCR phenotypes. Thus, GPCRs are the largest and most important class of cell surface membrane proteins, and they are involved in virtually all physiological processes.

GPCRs are naturally dynamic proteins that function to allosterically communicate signals from the extracellular surface to changes in intracellular function. Not surprisingly, these membrane proteins are one of the largest target classes for drug discovery and development. Understanding the molecular interactions that govern ligand affinity and regulation of protein function is a key requisite of structure-assisted drug discovery and development.

GPCRs transduce signals via different families of heterotrimeric G proteins, and other regulatory/scaffold proteins, exemplified by arrestins. The diversity in transducer engagement has led to a class of ligands termed biased agonists that activate different spectrums of signalling from the same receptor. Understanding the molecular basis for drug efficacy and biased agonism is also key for optimal development of therapeutics.

The ability to capture high resolution structure of GPCRs in different states, in complex with different transducer and other regulatory proteins, and to ligands of different pharmacology is an important challenge for structural biology and is a key focus of much of the research within the Centre.


Subproject 1: Understanding Receptor Activation and Transducer Coupling

We have extended the spectrum of GPCR states amenable to structural resolution in study of the CGRPR to provide understanding of the transition between apo receptor, ligand-bound receptor and ligand- and transducer-bound receptor, as well as integrating cryo-EM and HDX-MS to probe dynamics of different states.

We solved peptide liganded complexes of therapeutically important amylin and calcitonin receptors and revealed that distinct template peptides utilise distinct activation mechanisms, gaining insight from high-resolution cryo-EM structure and conformational dynamics of individual complexes. This work, along with pharmacological characterisation of different peptides acting at this receptor family, will form the basis for a collaborative project with Novo Nordisk.

We provided the first structures of a receptor (CCK1R) bound to Gs and Gq proteins providing insight into G protein selectivity across different receptor classes and modulation of ligand affinity and G protein selectivity, as well as the identification of a novel allosteric modulator of the CCK1R. This work is also contributing to the collaborative project with Astex Pharmaceuticals.

Subproject 2: Understanding the Molecular Basis for Biased Agonism

We are extending investigation into the molecular basis for biased agonism at the incretin peptide receptor family that has provided new insight into key atomic interactions and also the distinct dynamics of receptors with ligands of distinct pharmacology. We have projects in this space that are being developed collaboratively with Boehringer Ingelheim, and we are currently progressing discussions for a project with Servier.

Subproject 3: Allosteric and Bitopic Ligand Regulation of GPCRs

We have been pursuing pharmacological and structural studies with a focus on muscarinic and adenosine receptors. The work on the A1 adenosine receptor published in Nature revealed an unexpected site for allosteric ligand binding with the structural work linked to in vitro and in vivo pharmacological validation of the potential for targeting this site. A separate component of this research area is investigation of allosteric regulation of GPCRs through dimerization. A project in this latter space is being developed with Dimerix.

Subproject 4: Development of Biochemical Approaches for Determination of Inhibitor-bound GPCR Structures

The small size of apo and inactive GPCRs has made these particularly challenging for cryo-EM. We are currently exploring different methods, both biochemical and in cryo-EM data processing, to advance this area of research. We have collaborative projects in this space with Pfizer, Astex and Boehringer Ingelheim.

Subproject 5: Development of Approaches for Determination of Orphan GPCR Structures

Orphan GPCRs lack known natural ligands but in many cases have been implicated in disease. Generating structures of orphan receptors can potentially open up opportunities for structure-based design. We currently have collaborative projects with AstraZeneca and Boehringer Ingelheim in this research area.

Research Highlights

Structure and Dynamics of CGRP Receptor in Apo and Peptide-bound Forms

Research Fellow: Tracy Josephs

Josephs TM, et al. Science 372:eabf7258, 2021. doi:10.1126/ science.abf7258

Researchers from the Monash University Node have harnessed cutting-edge technology to discover the progression of molecular events that lead to migraine – something that, until now, has remained a mystery. The discovery has filled one of the most important gaps in our understanding of how migraines are activated. Published in the prestigious journal Science, the breakthrough study was led by a team of researchers from the Monash Institute of Pharmaceutical Sciences (MIPS) and the recently established ARC Centre for Cryo-EM of Membrane Proteins (CCeMMP). One of the most common causes for migraine is abnormal levels of activation of the target for an extremely potent vascular regulator, calcitonin gene-related peptide (CGRP).

The newest and most exciting treatments for migraine act by blocking this activity, but how CGRP activates its receptor at the molecular level has been poorly understood. In this study, the researchers applied cryo-electron microscopy (cryo-EM) to, for the first time, show how the binding of CGRP to the receptor leads to receptor activation that, in turn, leads to the onset of migraine. Until now this simply wasn’t possible as proteins such as the CGRP receptor were too small and too mobile to be captured and studied by any method.

Lead author of the study, ARC CCeMMP member Dr Tracy Josephs from MIPS said: “To really understand what triggers migraines, we need to be able to study structure and dynamics using unmodified forms of the receptor – this has been a major technical hurdle in understanding the progression of molecular events that link CGRP binding to activation of the cellular signalling pathways that govern migraine pain, and one the team have now overcome.” “Based on the structures and data from complementary biophysical techniques, we showed that initial binding of CGRP to the receptor caused unexpectedly small conformational changes in the most prevalent form of the receptor. It was the coordinated change in dynamics of the external (CGRP binding) face of the receptor and the intracellular face that was the key, and visualising this would not have been possible by other methods.”

Migraines affect approximately five million Australians. It is a neurological condition that can cause multiple, debilitating symptoms including intense headaches, nausea, vomiting, difficulty speaking, numbness or tingling and sensitivity to light and sound.

Professor Patrick Sexton, co-lead on the study and Director of the ARC CCeMMP said: “This is an example of the enormous benefits of fundamental basic research in addressing major unmet medical needs”.

The team from MIPS and ARC CCeMMP worked with collaborators from the University of Tokyo, the University of Otago and the Hudson Institute of Medical Research.

The data underpinning this work was also used in the “CCeMMP EMPIAR data processing challenge”

( that was run as an international outreach activity.

A Structural Basis for Amylin Receptor Phenotype

Research Fellow: Jason Cao

Cao J, et al. Science 375:eabm9609, 2022 doi:10.1126/science.abm9609.

Australian researchers have announced a discovery which ultimately could play a major role in reducing obesity. In a report published in the leading international research journal, Science, scientists from the Monash Institute of Pharmaceutical Sciences (MIPS) and the ARC Center for Cryo-EM of Membrane Proteins (CCeMMP), affiliated with Monash University, have pinpointed how a promising group of anti-obesity drugs known as DACRAs (dual amylin and calcitonin receptor agonists) activate various receptors in the body. Previously the activation process had not been fully understood, which limited the clinical advancement of this class of weight-loss drug. “Our work opens up opportunities for the design and development of more effective DACRA therapies that could be used to better treat the spectrum of overweight and obese patients, and those with related metabolic disease,” said Monash University Professor of Drug Discovery Biology and ARC CCeMMP director Patrick Sexton MIPS structural biologist doctoral candidate Jason Cao said the breakthrough was made by using cutting-edge technology called cryo-electron microscopy (cryo-EM). “To really understand how these drugs may work, we needed to visualize molecular level details of how the different types of template peptides were bound to each of the four different target receptors,” he said. Cao said cryo-EM enabled the scientists to “capture molecular details of the interactions that drive activity” along with “information on the dynamics of the protein that are critical to selectivity and function.”

The researchers said they were surprised by their findings because it had been assumed the “molecular architecture of the amylin receptors would dictate how the peptides worked.” Instead, they saw “dramatic differences in the conformations and dynamics of the individual receptors when bound to different peptides.”

The quest for such discoveries has become ever more urgent, with World Health Organizationestimating that by 2025, approximately 167 million people — adults and children — will become less healthy because they are overweight or obese. “The team has revealed structural details on how calcitonin and amylin receptors are activated” said Professor Wootten, co-lead on the study and leader of the Monash node of the ARC CCeMMP. “This work will support the development of next generation DACRA drugs as weapons to address the rising obesity epidemic”.

The team from MIPS and ARC CCeMMP worked with collaborators from the University of Tokyo, and the University of Otago.

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