Biology subjects

Briddon, S. J.

Publications and source records attributed to Briddon, S. J..

9 recordsLinked to original sources

bioRxiv2024

Agonist efficacy at the B2AR is driven by agonist-induced differences in receptor affinity for the Gs protein, not ligand binding kinetics.

IntroductionThe {beta}2-adrenoceptor ({beta}2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby {beta}2AR agonists relieve bronchoconstriction. The {beta}2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the {beta}2AR is not understood. We hypothesized that the kinetics of ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of {beta}2AR agonists and examined the correlation between ligand and mini-Gs binding kinetics and efficacy. MethodsWe used a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) based competition ligand binding assay to measure the affinity and residence times of a range of {beta}2AR agonists binding to the human {beta}2AR. TR-FRET between Lumi4-Tb3+ on the N terminus of the {beta}2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these {beta}2AR agonists to activate the heterotrimeric Gs protein was measured using the CASE Gs protein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the G subunit and Venus acceptor on the G{psi}, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised {beta}2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gs was used to dissociate the {beta}2AR-nLuc: Venus-mini-Gs complex. ResultsCharacterisation of the molecular pharmacology of seven {beta}2AR agonists showed a broad range of ligand binding affinities (pKi = 4.4 {+/-} 0.09 to 9.2 {+/-} 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log{tau} ) to activate the Gs protein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36{+/-}0.64x105 to 9.19{+/-} 0.42x105) and affinity (Kd) values of mini-Gs binding to the agonist-{beta}2AR complex (pKd =6.0 to 6.7). Both an increase in ligand driven mini-Gs kon(fast) rate and associated increase in mini-Gs pKd for the receptor, were moderately correlated with efficacy (log{tau} ) (R2 =0.58 and R2 =0.50 respectively). ConclusionsThese data support a model in which agonists of increased efficacy cause the {beta}2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support a role for agonist binding kinetics in the molecular basis of efficacy.

pharmacology and toxicology↗

bioRxiv2023

Key phosphorylation sites for robust β-arrestin2 binding at the mu-opioid receptor revisited

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/570073v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@198d7f5org.highwire.dtl.DTLVardef@18a3d9forg.highwire.dtl.DTLVardef@d3c9e8org.highwire.dtl.DTLVardef@6ca5f2_HPS_FORMAT_FIGEXP M_FIG C_FIG Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, we and others have shown that phosphorylation is important for {beta}-arrestin recruitment to the receptor, and that ligands of different efficacies induce distinct patterns, or barcodes, of receptor phosphorylation. Within the MOR C-tail, the 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser acts as a primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr residues can get subsequently phosphorylated. Here we used HEK293 GRK KO cells, in combination with phosphosite specific antibodies and site-directed mutagenesis of the MOR, to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and {beta}-arrestin2 recruitment. We show that both GRK subfamilies (GRK2/3 and GRK5/6) promote phosphorylation of Thr370 and Ser375. However, only GRK2/3 induce phosphorylation of Thr376 and Thr379, which is required to promote robust {beta}-arrestin recruitment to the receptor. Moreover, while DAMGO and fentanyl can engage all kinases to promote Thr370 and Ser375 phosphorylation, under endogenous GRK expression conditions, morphine-induced phosphorylation of these residues is specifically mediated by GRK5/6. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of the different GRK subfamilies plays an important role in determining the tissue responses of distinct opioid agonists.

pharmacology and toxicology↗

bioRxiv2023

A time-resolved Forster resonance energy transfer assay to investigate inhibitor binding to ABCG2

The human ATP-binding cassette (ABC) transporter, ABCG2 is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Forster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps. HighlightsO_LIABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitors C_LIO_LIWe introduce a time-resolved Forster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2 C_LIO_LIWe confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor C_LI

biochemistry↗

bioRxiv2021

Simple methods for quantifying super-resolved cortical actin

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or corrals, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by both Super Resolved Radial Fluctuations (SRRF) and expansion microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31{micro}m2 {+/-} 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

cell biology↗

bioRxiv2021

Acylation of the incretin peptide exendin-4 directly impacts GLP-1 receptor signalling and trafficking

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to promote albumin binding in order to prolong their pharmacokinetics, but the potential for these ligand changes to influence GLP-1R signalling has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). In this work we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16, for their relative propensities to recruit and activate G proteins and {beta}-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes. Both ligands had similar cAMP potency but the exendin-4-C16 showed [~]2.5-fold bias towards G protein recruitment and a [~]60% reduction in {beta}-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with a reduced ability to promote the movement of the GLP-1R extracellular domain, as determined using a conformational biosensor approach, and a [~]70% increase in insulin secretion. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Overall, our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. Significance statementAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.

pharmacology and toxicology↗

bioRxiv2020

Functional solubilisation of the β2-adrenoceptor (β2AR) using Diisobutylene maleic acid (DIBMA).

The β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical GPCR for biophysical studies. Solubilisation of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilises membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilise membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β2AR in the polymer Diisobutylene maleic acid (DIBMA). We demonstrate that β2AR remains functional in the DIBMA lipid particle (DIBMALP) and shows improved thermal stability compared to the n-Dodecyl-β-D-Maltopyranoside (DDM) detergent solubilised β2AR. This unique method of extracting β2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilising mutations and the use of detergents, particularly for functional biophysical studies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

pharmacology and toxicology↗

bioRxiv2020

Ligand-directed covalent labelling of a GPCR with a fluorescent tag

Here, we describe rational design of a compound that covalently and selectively labels a G protein-coupled receptor (GPCR) in living cells with a fluorescent moiety. Using wild-type adenosine A2A receptor as a model system, we show fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

pharmacology and toxicology↗

bioRxiv2019

Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is essential for embryonic development and impacts on diseases such as cancer, Alzheimers and inflammatory diseases. ADAM10 is a molecular scissor that proteolytically cleaves the extracellular region from over 100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors and chemokines. ADAM10 was recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function is critically dependent on a TspanC8 partner. To address this, we generated the first monoclonal antibodies to Tspan15 as a model TspanC8. These were used to show that ADAM10 is the principal Tspan15-interacting protein, that Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Together these findings suggest that ADAM10 exists as an intimate ADAM10/TspanC8 scissor complex.

cell biology↗