Generation and characterization of nanobodies targeting GPCR.

G protein-coupled receptors (GPCRs) are a large family of cell membrane proteins that are important targets for drug discovery. Nanobodies, also known as VHH (variable domains of heavy chain-only antibodies, HcAbs) antibodies, are small antibody fragments derived from camelids that have gained significant attention as potential therapeutics for targeting GPCRs due to their advantages over conventional antibodies. However, there are challenges in developing nanobodies targeting GPCRs, among which epitope accessibility is the most significant because the cell membrane partially shields the GPCR surface. We developed a universal protocol for making nanobodies targeting GPCRs using the cell membrane extract of GPCR-overexpressing HEK293 cells as the llama/alpaca immunization antigen. We constructed an immune VHH library and identified nanobodies by phage display bio-panning. The monoclonal nanobodies were recombinantly expressed in Escherichia coli (E. coli) and purified to characterize their binding potency.

Human GLP1R variants affecting GLP1R cell surface expression are associated with impaired glucose control and increased adiposity.

The glucagon-like peptide 1 receptor (GLP1R) is a major drug target with several agonists being prescribed in individuals with type 2 diabetes and obesity1,2. The impact of genetic variability of GLP1R on receptor function and its association with metabolic traits are unclear with conflicting reports. Here, we show an unexpected diversity of phenotypes ranging from defective cell surface expression to complete or pathway-specific gain of function (GoF) and loss of function (LoF), after performing a functional profiling of 60 GLP1R variants across four signalling pathways. The defective insulin secretion of GLP1R LoF variants is rescued by allosteric GLP1R ligands or high concentrations of exendin-4/semaglutide in INS-1 823/3 cells. Genetic association studies in 200,000 participants from the UK Biobank show that impaired GLP1R cell surface expression contributes to poor glucose control and increased adiposity with increased glycated haemoglobin A1c and body mass index. This study defines impaired GLP1R cell surface expression as a risk factor for traits associated with type 2 diabetes and obesity and provides potential treatment options for GLP1R LoF variant carriers.

Conformational Dynamics of the Activated GLP-1 Receptor-Gs Complex Revealed by Cross-Linking Mass Spectrometry and Integrative Structure Modeling.

Despite advances in characterizing the structures and functions of G protein-coupled receptors (GPCRs), our understanding of GPCR activation and signaling is still limited by the lack of information on conformational dynamics. It is particularly challenging to study the dynamics of GPCR complexes with their signaling partners because of their transient nature and low stability. Here, by combining cross-linking mass spectrometry (CLMS) with integrative structure modeling, we map the conformational ensemble of an activated GPCR-G protein complex at near-atomic resolution. The integrative structures describe heterogeneous conformations for a high number of potential alternative active states of the GLP-1 receptor-Gs complex. These structures show marked differences from the previously determined cryo-EM structure, especially at the receptor-Gs interface and in the interior of the Gs heterotrimer. Alanine-scanning mutagenesis coupled with pharmacological assays validates the functional significance of 24 interface residue contacts only observed in the integrative structures, yet absent in the cryo-EM structure. Through the integration of spatial connectivity data from CLMS with structure modeling, our study provides a new approach that is generalizable to characterizing the conformational dynamics of GPCR signaling complexes.

Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface.

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine-tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAMs tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

Anti-ATR001 monoclonal antibody ameliorates atherosclerosis through beta-arrestin2 pathway.

BACKGROUND: Our previous study developed ATRQß-001 vaccine, which targets peptide ATR001 from angiotensin Ⅱ (Ang Ⅱ) receptor type 1 (AT1R). The ATRQß-001 vaccine could induce the production of anti-ATR001 monoclonal antibody (McAb-ATR) and inhibit atherosclerosis without feedback activation of the renin-angiotensin system (RAS). This study aims at investigating the underexploited mechanisms of McAb-ATR in ameliorating atherosclerosis. METHODS: AT1R-KO HEK293T cell lines were constructed to identify the specificity of McAb-ATR and key sites of ATRQß-001 vaccine. Beta-arrestin1 knock-out (Arrb1-/-) mice, Beta-arrestin2 knock-out (Arrb2-/-) mice, and low-density lipoprotein receptor knock-out (LDLr-/-) mice were used to detect potential signaling pathways affected by McAb-ATR. The role of McAb-ATR in beta-arrestin and G proteins (Gq or Gi2/i3) signal transduction events was also investigated. RESULTS: McAb-ATR could specifically bind to the Phe182-His183-Tyr184 site of AT1R second extracellular loop (ECL2). The anti-atherosclerotic effect of McAb-ATR disappeared in LDLr-/- mice transplanted with Arrb2-/- mouse bone marrow (BM) and BM-derived macrophages (BMDMs) from Arrb2-/- mice. Furthermore, McAb-ATR inhibited beta-arrestin2-dependent extracellular signal regulated kinase1/2 (ERK1/2) phosphorylation, and promoted beta-arrestin2-mediated nuclear factor kappa B p65 (NFκB p65) inactivity. Compared with conventional AT1R blockers (ARBs), McAb-ATR did not inhibit Ang Ⅱ-induced uncoupling of heterotrimeric G proteins (Gq or Gi2/i3) and Gq-dependent intracellular Ca2+ release, nor cause RAS feedback activation. CONCLUSIONS: Through regulating beta-arrestin2, McAb-ATR ameliorates atherosclerosis without affecting Gq or Gi2/i3 pathways. Due to high selectivity for AT1R and biased interaction with beta-arrestin2, McAb-ATR could serve as a novel strategy for treating atherosclerosis.