ORCID Profile
0000-0001-6332-2793
Current Organisation
Florey Institute of Neuroscience and Mental Health
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Biomolecular Modelling and Design | Biochemistry and Cell Biology | Receptors and Membrane Biology | Structural Biology (incl. Macromolecular Modelling) |
Nervous System and Disorders | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences
Publisher: Wiley
Date: 05-2005
Abstract: Recent molecular and pharmacologic studies have identified LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family, as a cognate receptor for insulin-like peptide-3 (INSL3). LGR8 mRNA has been detected in various tissues, but the precise roles of these INSL3-LGR8 systems are unknown. In this study we first investigated the presence and cellular localization of LGR8 mRNA in both adult and developing rat kidney and subsequently examined the possible role of INSL3-LGR8 signaling in cultured mesangial cells. LGR8 mRNA was detected in the kidney by polymerase chain reaction and localized by in situ hybridization in mature glomerular mesangial cells within the renal cortex, with highest levels detected at embryonic day 18 and lowest levels in adult kidney. Synthetic INSL3 inhibited the proliferation of mesangial cells in primary culture, indicating the presence of functional LGR8 on these cells. These findings suggest that INSL3/LGR8 signaling may be involved in the genesis and/or developmental maturation of renal glomeruli and in the regulation of mesangial cell density in the adult kidney.
Publisher: Wiley
Date: 05-2005
Abstract: LGR8 was recently identified as a cognate receptor for insulin-like peptide-3 (INSL3), and INSL3-LGR8 signaling is best known for its role in testis descent during development. LGR8 mRNA has been detected in various human tissues including brain, but the regional and cellular distribution of LGR8 expression in the mammalian central nervous system is unknown. Therefore, in this study we investigated the presence and localization of LGR8 mRNA in rat brain using reverse transcription-polymerase chain reaction and in situ hybridization histochemistry. Results revealed a distinct distribution of LGR8 in forebrain, with transcripts principally restricted to the posterior thalamus and highest densities detected in the parafascicular nucleus of both adult and developing rats. Unexpectedly, INSL3 mRNA was not detected in brain by similar methods, but preliminary electrophysiologic studies of parafascicular neurons revealed that INSL3 altered their activity. These findings suggest that LGR8 signaling may be involved in sensorimotor control in the rat and perhaps other species, particularly via actions on parafascicular neurons that project to basal ganglia and are depleted in Parkinson's disease.
Publisher: Elsevier BV
Date: 10-2020
Publisher: The Endocrine Society
Date: 11-2012
DOI: 10.1210/ME.2012-1188
Publisher: Wiley
Date: 18-08-2020
Abstract: The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α 1A ‐AR and α 1B ‐AR, which belong to the G protein‐coupled receptor (GPCR) superfamily, by employing the solution‐based ligand‐observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype‐selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α 1A ‐AR‐selective agonist A‐61603 relative to an endogenous agonist, epinephrine, at both α 1A ‐AR and α 1B ‐AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back‐calculated spectra based on ligand poses obtained from all‐atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of ( R )‐A‐61603 towards α 1A ‐AR, thus demonstrating an approach for targeting subtype selectivity in ARs.
Publisher: Oxford University Press (OUP)
Date: 13-08-2018
DOI: 10.1093/BRAIN/AWY209
Abstract: Baker, Gordon et al. present the first international case series describing the neurodevelopmental disorder associated with Synaptotagmin 1 (SYT1) de novo missense mutations. Key features include movement abnormalities, severe intellectual disability, and hallmark EEG alterations. Expression of patients’ SYT1 mutations in mouse neurons disturbs presynaptic vesicle dynamics in a mutation-specific manner.
Publisher: Elsevier BV
Date: 05-2020
Publisher: Elsevier BV
Date: 04-2022
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03950.X
Abstract: The primary binding sites of the relaxin and insulin-like peptide 3 (INSL3) receptors, RXFP1 and RXFP2, are found within the leucine-rich repeats (LRRs) of the ectodomains. Specific B-chain residues in the peptides interact with residues in the inner beta-sheets of the LRRs of the receptors. Relaxin binds to RXFP2 with high affinity, although INSL3 has a very poor affinity for RXFP1. In this paper we present evidence that relaxin binds to the LRRs of RXFP2 in a manner similar to INSL3 binding to its receptor. Additionally, we introduce a model of this binding interaction and compare it to an alternate model for relaxin-RXFP1 binding.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Wiley
Date: 05-2005
Abstract: Rodent models have been used for many years to probe the actions of relaxin. Identification of the orthologs of human leucine-rich repeat-containing g-protein-coupled receptor 7 (LGR7), the relaxin receptor, in mouse and rat will enable characterization of the response of LGR7 to relaxin in these species. Partial LGR7 homologous sequences from mouse and rat were discovered in the Celera and NCBI gene databases, lified, cloned, and sequenced. At the protein level, mouse and rat LGR7 are 85.2% and 85.7% identical to human LGR7. Mouse and rat LGR7 were able to bind to and be activated by relaxin ligands.
Publisher: Elsevier BV
Date: 10-2021
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.PHARMTHERA.2018.02.008
Abstract: The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.
Publisher: Wiley
Date: 26-12-2018
DOI: 10.1002/PRP2.455
Publisher: Wiley
Date: 05-2005
Abstract: The relaxin receptor (LGR7) and the insulin-like peptide 3 (INSL3) receptor (LGR8) are unique LGR family members in possessing a single, functionally important amino terminal LDL-A module.1 Mouse and rat cDNA was screened for LGR7 and LGR7 splice variant expression. A uterus-specific exon 4 deleted variant was identified and named LGR7-Truncate. Deletion of exon 4 results in a premature stop codon and a transcript that putatively encodes a secreted protein containing LGR7's LDL-A module. Expression of LGR7-Truncate with LGR7 in HEK-293T cells resulted in decreased relaxin-induced signaling of LGR7. LGR7-Truncate is potentially an endogenous regulator of LGR7 signaling.
Publisher: Elsevier BV
Date: 11-2006
Publisher: Wiley
Date: 05-2005
Abstract: Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.
Publisher: Frontiers Media SA
Date: 2013
Publisher: Elsevier BV
Date: 11-2020
Publisher: Elsevier BV
Date: 04-2022
Publisher: Elsevier BV
Date: 2023
Publisher: Springer Science and Business Media LLC
Date: 23-03-2022
DOI: 10.1038/S42003-022-03191-5
Abstract: Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB 5 toxin, inhibits Gα i/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gα z . The role of Gα z signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB 5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gα i/o and Gα z G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein.
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.BBAMEM.2014.07.015
Abstract: The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening c aigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.
Publisher: Springer Science and Business Media LLC
Date: 18-04-2016
DOI: 10.1038/NCOMMS11344
Abstract: H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.
Publisher: Elsevier BV
Date: 06-2013
DOI: 10.1016/J.CBPA.2013.04.002
Abstract: Integral membrane proteins (IMPs) are crucial components of all cells but are difficult to study in vitro because they are generally unstable when removed from their native membranes using detergents. Despite the major biomedical relevance of IMPs, less than 1% of Protein Data Bank (PDB) entries are IMP structures, reflecting the technical gap between studies of soluble proteins compared to IMPs. Stability can be engineered into IMPs by inserting stabilizing mutations, thereby generating proteins that can be successfully applied to biochemical and structural studies when solubilized in detergent micelles. The identification of stabilizing mutations is not trivial, and this review will focus on the methods that have been used to identify stabilized membrane proteins, including alanine scanning and screening, directed evolution and computational design.
Publisher: The Royal Society of Chemistry
Date: 17-08-2022
DOI: 10.1039/9781839165702-00346
Abstract: G protein-coupled receptors are the largest family of integral membrane proteins in humans that have roles in almost all physiological processes. The binding of extracellular ligands allosterically modulates the intracellular interaction of the GPCR with transducer proteins such as G proteins and arrestins. This allosteric coupling operates via a network of conserved microswitches to adjust the equilibrium of active, intermediate and inactive states of the GPCR. Crystallography and cryo-electron microscopy have determined the structures of many active and inactive state GPCRs, while solution-state methods such as NMR spectroscopy inform on the dynamics of additional states and their role in signalling. In addition, solution NMR spectroscopy is providing insight into the pathways and mechanisms of ligand binding, including disordered peptides, to GPCRs. This chapter reviews the challenges in preparing GPCRs for solution NMR data collection, the knowledge gained about the conformational landscapes and ligand binding to GPCRs.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2023
DOI: 10.1038/S41467-023-38894-8
Abstract: The neurotensin receptor 1 (NTS 1 ) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS 1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13 C ε H 3 -methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS 1 :G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.
Publisher: Proceedings of the National Academy of Sciences
Date: 22-01-2014
Abstract: Only a tiny fraction ( %) of the unique structures in the protein database correspond to membrane proteins, and only a few of these are of eukaryotic origin, representing potential drug targets. The difficulties in structure determination of these proteins are due to two specific complications, which are unique for membrane proteins: first, low expression levels and, second, the necessity for detergent micelles, which are often destabilizing as they mimic the hydrophobic membrane environment only poorly. We prove that directed evolution has the potential to overcome these problems by determining several structures of evolved eukaryotic G protein–coupled receptor variants. High functional expression levels and superior receptor stability in harsh detergents allowed us to gain deeper insights into this important receptor family.
Publisher: Bentham Science Publishers Ltd.
Date: 10-2005
Publisher: Springer Science and Business Media LLC
Date: 12-01-2018
DOI: 10.1038/S41598-017-18045-Y
Abstract: Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/ In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
Publisher: Wiley
Date: 11-2004
DOI: 10.1111/J.1440-1681.2004.04075.X
Abstract: 1. Relaxin is an extracellular matrix (ECM)-remodelling hormone that is functionally important in reproductive tissues, brain, lung and heart. 2. Recently, the human relaxin receptor was identified as leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7). 3. Using human LGR7 as a template, we identified mouse and rat LGR7 orthologues in the Celera and National Centre for Biotechnology Information databases. 4. At the protein level, mouse and rat LGR7 share 85.2 and 85.7% identity with human LGR7, respectively. 5. Mouse LGR7 mRNA was detected in all tissues where relaxin binding is observed. 6. Mouse and rat LGR7 bound [33P]-relaxin with high affinity and, upon relaxin treatment, both receptors stimulated cAMP production in transfected HEK 293T cells. 7. These results indicate that mouse and rat LGR7 are the relaxin receptors in these species. 8. The actions of relaxin in rodents are well characterized, providing an established platform for research into the molecular pharmacology of the highly conserved relaxin receptor.
Publisher: The Endocrine Society
Date: 07-2007
DOI: 10.1210/ME.2007-0097
Abstract: The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [125I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/BF02442569
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3LC00043E
Abstract: The developed acoustofluidic device realizes microparticle and cell patterning with acoustophoretic force. It combines a planar acoustic resonator and a microstructured surface which provides complex particle formations with sub-wavelength precision.
Publisher: American Chemical Society (ACS)
Date: 14-03-2202
DOI: 10.1021/ACSCHEMBIO.8B00191
Abstract: α
Publisher: Springer Science and Business Media LLC
Date: 24-04-2017
DOI: 10.1038/S41598-017-01227-Z
Abstract: G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS 1 ). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS 1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS 1 showed non-competitive binding to the peptide ligand neurotensin8–13 when compared to E. coli produced en2NTS 1 . 1 H- 13 C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13 CH 3 -methionine labelled en2NTS 1 . The results obtained, indicate CFPS produced en2NTS 1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.
Publisher: Elsevier BV
Date: 10-2018
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03955.X
Abstract: The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and activation, together with advances in methods for structural characterization, has enabled the key structural features of the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarities in primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the four known relaxin family peptide receptors (RXFP1 to -4). Most of our knowledge on structure and function relates to the relaxin-RXFP1, insulin-like peptide 3 (INSL3)-RXFP2, and relaxin-3-RXFP3 systems, with information accumulating not only on the critical ligand structures but also the domains and residues on the receptor itself that are required for specificity and activation. These studies provide the framework for the design of small-molecule mimetics. While the B-chain cassette R-X-X-X-R-X-X-I, defined by Büllesbach and Schwabe, is essential for binding and activation of RXFP1, it is now recognized that the A chain, particularly the N-terminal domain, is also critical for receptor specificity. Studies of the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists. The relaxin-3 A chain-INSL5 B chain chimeric peptide and analogs with C-terminal truncations of the B chain, developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are proving invaluable for in vivo studies of the relaxin-3-RXFP3 system.
Publisher: Elsevier BV
Date: 06-2023
Publisher: Springer Science and Business Media LLC
Date: 29-11-2021
DOI: 10.1186/S13064-021-00156-Y
Abstract: Myelination is a highly regulated process in the vertebrate central nervous system (CNS) whereby oligodendrocytes wrap axons with multiple layers of insulating myelin in order to allow rapid electrical conduction. Establishing the proper pattern of myelin in neural circuits requires communicative axo-glial interactions, however, the molecular interactions that occur between oligodendrocytes and axons during developmental myelination and myelin maintenance remain to be fully elucidated. Our previous work identified G protein-coupled receptor 62 (Gpr62), an uncharacterized orphan g-protein coupled receptor, as being selectively expressed by mature oligodendrocytes within the CNS, suggesting a potential role in myelination or axoglial interactions. However, no studies to date have assessed the functional requirement for Gpr62 in oligodendrocyte development or CNS myelination. To address this, we generated a knockout mouse strain lacking the Gpr62 gene. We assessed CNS myelination during both postnatal development and adulthood using immunohistochemistry, electron microscopy and western blot. In addition, we utilized AAV-mediated expression of a tagged Gpr62 in oligodendrocytes to determine the subcellular localization of the protein in vivo. We find that virally expressed Gpr62 protein is selectively expressed on the adaxonal myelin layer, suggestive of a potential role for Gpr62 in axo-myelinic signaling. Nevertheless, Gpr62 knockout mice display normal oligodendrocyte numbers and apparently normal myelination within the CNS during both postnatal development and adulthood. We conclude that in spite of being well-placed to mediate neuronal-oligodendrocyte communications, Gpr62 is overall dispensable for CNS myelination.
Publisher: Wiley
Date: 30-07-2019
DOI: 10.1002/PRP2.513
Publisher: Elsevier BV
Date: 2018
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9121-1_3
Abstract: Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete
Publisher: Cold Spring Harbor Laboratory
Date: 25-07-2021
DOI: 10.1101/2021.07.21.21260857
Abstract: Synaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioural disturbance and EEG abnormalities. Here, we expand the genotypes and phenotypes and identify discriminating features of this disorder. We describe 22 in iduals with 15 de novo missense SYT1 variants. Evidence for pathogenicity is discussed, including ACMG criteria, known structure-function relationships, and molecular dynamics simulations. Quantitative behavioural data for 14 cases are compared to other monogenic neurodevelopmental disorders. Four variants lie in the C2A domain with the remainder in the C2B. We classify 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes include delayed developmental milestones, ophthalmic problems, abnormal EEG, movement disorders and sleep disturbance. Discriminating behavioural characteristics were severity of motor and communication impairment, presence of motor stereotypies and mood instability. SYT1 variants associated with neurodevelopmental disorder extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severity than initially reported. This work guides diagnosis and molecular understanding of this rare neurodevelopmental disorder, and highlights a key role for SYT1 function in emotional regulation, motor control and emergent cognitive function.
Publisher: American Chemical Society (ACS)
Date: 29-04-2020
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.BBAMEM.2018.03.020
Abstract: Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS
Publisher: Wiley
Date: 04-11-2014
DOI: 10.1002/BIT.25451
Abstract: Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link in idual members of erse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from erse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS.
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03949.X
Abstract: The receptors for relaxin and insulin-like peptide 3 (INSL3) are now well-characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G-protein-coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine-rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low-density lipoprotein class A (LDL-A) module at their N-terminus. Ligand-mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL-A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL-A module is currently unknown. The specific understanding of the mechanisms underlying ligand-mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.
Publisher: Elsevier BV
Date: 2018
Publisher: Springer Science and Business Media LLC
Date: 2002
Publisher: Springer Science and Business Media LLC
Date: 12-06-2017
DOI: 10.1038/S41598-017-03638-4
Abstract: Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.
Publisher: Elsevier BV
Date: 09-2006
Publisher: American Chemical Society (ACS)
Date: 06-06-2008
DOI: 10.1021/BI800535B
Abstract: The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at in idual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.
Publisher: Elsevier BV
Date: 02-2012
DOI: 10.1016/J.JMB.2012.11.015
Abstract: G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million in idual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies.
Publisher: MDPI AG
Date: 08-02-2022
DOI: 10.3390/IJMS23031908
Abstract: Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.
Publisher: Bioscientifica
Date: 05-2006
DOI: 10.1677/JOE.1.06697
Abstract: Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3–LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125 I-labelled human INSL3-binding sites and the nature of INSL3–LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125 I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3–LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.
Publisher: Springer Science and Business Media LLC
Date: 19-01-2022
DOI: 10.1038/S41467-021-27911-3
Abstract: α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and h ered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α 1B AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α 1B AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α 1B AR with α 2 ARs, and by constructing α 1B AR-α 2C AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α 1B AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
Location: Australia
Start Date: 2021
End Date: 2012
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2009
End Date: 2012
Funder: Swiss National Science Foundation
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2008
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2019
End Date: 2022
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 2021
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2018
End Date: 2021
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 10-2019
End Date: 12-2022
Amount: $464,233.00
Funder: Australian Research Council
View Funded Activity