G protein-coupled receptors (GPCRs) are membrane proteins that act as signal transducers across cell membranes. Class B1 GPCRs, a subset of 15 receptors activated by peptide hormones, are involved in important physiological processes and diseases, making them a popular target for drug development. GPCRs are dynamic proteins and can adopt a myriad of conformational states, allowing them to bind and activate multiple intracellular signal transducers, including G proteins and β-arrestins, though all class B1 GPCRs primarily couple to the stimulatory G protein (Gs). Cryogenic electron microscopy (cryo-EM) structures of all class B1 GPCRs bound to Gs are available and provide meaningful insights into receptor function. However, there is a dearth of structural information on class B1 GPCRs in inactive and intermediate states or bound to other signal transducers, meaning we are currently only afforded a small vista into the conformational landscape these GPCRs sample. As cryo-EM-based 3D reconstructions are heavily dependent on protein stability and conformational homogeneity, currently available structures are largely limited to only those most stable conformations (i.e. Gs complexes). The present review focuses on technical aspects of obtaining class B1 GPCR structures using cryo-EM and new in silico methods that allow insight into unseen GPCR conformations, revealing more structural details of the conformational landscape.

G protein-coupled receptors (GPCRs) are key regulators of intercellular communication. One sub-family, named class B1 GPCRs, including the pituitary adenylate cyclase-activating polypeptide 1 receptor (PAC1R), is important for metabolic, cardiovascular, and endocrine functions. Currently, there is a wealth of structural data describing the active G protein-coupled state of class B1 GPCRs, and more limited information on inactive or intermediate states. Although these structures provide insight into conformational differences between different activation states, the receptor dynamics underpinning them remain largely unknown. Here, we employed hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the dynamics of PAC1R across three distinct states: inactive (apo), intermediate (peptide agonist-bound), and fully active (peptide agonist and G protein-bound). This revealed dynamics of different states along the PAC1R activation pathway and deepens the understanding of class B1 GPCR dynamics and molecular mechanisms of receptor activation.

Activation of thyroid-stimulating hormone receptor (TSHR) by autoantibodies is the primary cause of the autoimmune diseases Graves' hyperthyroidism and Graves' ophthalmopathy. Thus, recent pharmaceutical research has focused on blocking pathogenic TSHR activation. While this has potential to offer first-in-class disease-modifying medications to treat Graves' disease, the success of targeting drugs requires understanding of how TSHR-stimulating autoantibodies activate the receptor and mediate signalling. This study comprehensively characterized in vitro pharmacological profiles of two human TSHR-stimulating autoantibodies, M22 and K1-18, relative to bovine TSH (bTSH) in a HEK293A cell background expressing recombinant human TSHR. M22 and K1-18 exhibited subtle, G protein-specific, differences in activating Gα proteins, with both antibodies generally less potent and/or efficacious than bTSH. While both antibodies were equivalent in activation of most G proteins, K1-18 was less potent than M22 in activation of Gαs-short and Gαz. Interestingly, bTSH, M22 and K1-18 more potently activated Gα15 compared to other Gα proteins. In addition, M22 and K1-18 activated Gαs-dependent cAMP production and Gαq-coupled signalling (IP1 accumulation and iCa2+ mobilisation), recruited β-arrestin 1 and 2, and stimulated receptor internalization to early endosomes. K1-18 was less potent than bTSH in all these assays, while M22 was less potent than bTSH in iCa2+ mobilisation and β-arrestin recruitment. While M22 and K1-18 largely mirror the pharmacology of bTSH, quantification of revealed relative to bTSH they exhibit bias towards Gα12 activation, and away from β-arrestin 2 recruitment and iCa2+ mobilization relative to Gs and cAMP. Thus, these findings offer insight into the action of TSHR-stimulating antibodies.

Receptor activity-modifying proteins (RAMPs) are critical modulators of class B1 G protein-coupled receptors (GPCRs), altering receptor pharmacology, trafficking, and signaling. The calcitonin receptor (CTR) forms heterodimers with each of the three RAMPs to generate amylin receptors (AMYRs) with distinct agonist selectivity and signaling profiles. Although recent cryo-electron microscopy (cryoEM) structures have advanced our understanding of AMYR architecture in fully active states, the dynamic and mechanistic basis of RAMP-dependent modulation of the CTR remains poorly understood. Here, we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the conformational dynamics of the CTR alone and in complex with each RAMP in the apo (ligand-free) state. Our results reveal that RAMPs differentially influence the flexibility of key CTR domains, including the extracellular domain, transmembrane helices, and intracellular regions involved in G protein engagement. Furthermore, the RAMPs exhibit subtype-specific dynamic signatures, particularly within their transmembrane and C-terminal regions. Together, these findings reveal how RAMPs allosterically shape CTR conformational landscapes, providing a dynamic framework that links insights from static structural models to functional pharmacology.

G protein-coupled receptors (GPCRs) mediate information transfer to cells from the surrounding environment. In most cases, signaling is initiated or amplified when the receptor binds to an agonist, an event that alters the conformational profile of the receptor. Signal transduction results from interaction between the agonist-receptor complex and cytosolic partners such as G proteins, GPCR kinases (GRKs), and β-arrestins. Changes in agonist structure can lead to "signal bias", i.e., changes in the relative strength of signaling involving different partners. Some GPCRs, including those activated by long peptide hormones, continue to signal after internalization. In these cases, changes in agonist structure can lead to changes in the relative extent of signaling from different sites, e.g., cell surface vs endosomes ("location bias"). Many GPCRs are targets of approved drugs or drug candidates, and tuning signal bias and/or location bias is widely considered to be important for optimizing therapeutic profiles. Here we report another mechanism of modulating outcome via agonist modification: alteration of intracellular trafficking. The synthetic peptide agonist designated SPT, which contains five β-amino acid residues, was previously shown to activate the parathyroid hormone receptor-1 (PTH1R) and cause prolonged signaling in mice by an unknown mechanism. The SPT-PTH1R complex continues to stimulate cAMP production after internalization. We now find that the SPT-PTH1R complex impairs the sorting of early endosomes into recycling endosomes relative to the receptor complexed to the drug teriparatide. These findings suggest that altering intracellular GPCR trafficking patterns represents an unappreciated strategy for achieving prolonged action in vivo.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable, with GLP1R gene variation potentially linked to therapeutic outcomes. A GLP1R natural missense variant, A316T, protects against T2D and cardiovascular disease. Here, we generated and characterized a human GLP1R A316T mouse model. Human GLP1RA316T/A316T mice displayed lower fasting blood glucose versus wild-type littermates even under metabolic stress, as well as slower weight gain and alterations in islet cytoarchitecture, glucagon secretion, and liver metabolism under a high-fat, high-sucrose diet. This was however associated with blunted responses to pharmacological GLP-1RAs in vivo. Further investigations in β cell models demonstrated that human GLP1R A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies.

G-protein-coupled receptors (GPCRs) have key roles in physiology and are central targets for drug discovery and development1,2, but the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic3-6. Here we describe de novo design methods and a high-throughput receptor-diversion microscopy-based screen for generating GPCR-binding miniproteins with high affinity, potency and selectivity. We design miniprotein agonists that activate receptors involved in itch and pain, as well as antagonists that inhibit receptors implicated in cancer, metabolic disorders such as diabetes and obesity, and migraines. The cryo-electron microscopy (cryo-EM) structures of five receptor-bound designs are close to the computational design models. A designed chemokine receptor antagonist mobilizes haematopoietic stem and progenitor cells in vivo at a level comparable to a clinically used drug, with fewer adverse effects.

Polymer nanodiscs are a research tool that allows membrane proteins (MPs) to be encapsulated by a surrounding amphipathic polymer, isolated and studied to understand their structural and physiological properties. An advantage of using polymer nanodiscs over other membrane mimetics can be found in their ability to natively solubilise membrane proteins (MPs) within an annulus of cellular phospholipids, however, potential polymer interactions with membrane constituents can hinder MP activity making the selection of a suitable polymer critical. This work demonstrates the native solubilisation of G-protein coupled A2A adenosine receptor (A2AR) by polymers with alternating units and cationic charge, poly(N-methyl-4-vinyl pyridinium iodide-co-N-alkyl-maleimides) (poly(MVP-co-AlkylMs)), and novel statistical copolymers with pseudozwitterionic charge, poly(potassium 3-sulfopropyl methacrylate-co-2-(trimethyl-amino) ethyl methacrylate-co-n-butyl methacrylate) (poly(KSPMA-co-TMAEMA-co-BMA)), both synthesised using RAFT polymerisation. After surveying a library of polymers within each class, A2AR extraction was the most efficient using poly(MVP-co-BM) (1 : 1 MVP : BM) and poly(KSPMA-co-TMAEMA-co-BMA) (1 : 1 : 1 KSPMA : TMAEMA : BMA). The optimal pH, temperature, solubilisation time, polymer concentration and ionic strength conditions required for extracting A2AR were identified and enabled a large-scale A2AR-nanodisc preparation. The yield of A2AR-poly(MVP-co-BM) was superior to A2AR-poly(KSPMA-co-TMAEMA-co-BMA) nanodiscs after affinity purification. Functional assessment of the reconstituted receptors was undertaken using fluorescence correlation spectroscopy (FCS) to determine the ligand binding capacity of A2AR stabilised within an alternating cationic poly(MVP-co-BM). These native nanodiscs retained their ability to specifically bind A2AR ligand antagonists.

G protein-coupled receptors (GPCRs) play key roles in physiology and are central targets for drug discovery and development, yet the design of protein agonists and antagonists has been challenging as GPCRs are integral membrane proteins and conformationally dynamic. Here we describe computational de novo design methods and a high throughput "receptor diversion" microscopy-based screen for generating GPCR binding miniproteins with high affinity, potency and selectivity, and the use of these methods to generate MRGPRX1 agonists and CXCR4, GLP1R, GIPR, GCGR and CGRPR antagonists. Cryo-electron microscopy data reveals atomic-level agreement between designed and experimentally determined structures for CGRPR-bound antagonists and MRGPRX1-bound agonists, confirming precise conformational control of receptor function. Our de novo design and screening approach opens new frontiers in GPCR drug discovery and development.

Obesity is a major and increasingly prevalent chronic metabolic disease with numerous comorbidities. While recent incretin-based therapies have provided pharmaceutical inroads into treatment of obesity, there remains an ongoing need for additional medicines with distinct modes of action as independent or complementary therapeutics. Among the most promising candidates, supported by phase 1 and 2 clinical trials, is cagrilintide, a long-acting amylin and calcitonin receptor agonist. As such, understanding how cagrilintide functionally engages target receptors is critical for future development of this target class. Here, we determine structures of cagrilintide bound to Gs-coupled, active, amylin receptors (AMY1R, AMY2R, AMY3R) and calcitonin receptor (CTR) and compare cagrilintide interactions and the dynamics of receptor complexes with previously reported structures of receptors bound to rat amylin, salmon calcitonin or recently developed amylin-based peptides. These data reveal that cagrilintide has an amylin-like binding mode but, compared to other peptides, induces distinct conformational dynamics at calcitonin-family receptors that could contribute to its clinical efficacy.

Signal duration and subcellular location are emerging as important facets of G protein–coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cyclic adenosine monophosphate (cAMP) upon activation by the native hormone, GLP-1. cAMP production continues after the hormone–receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single β-amino acid substitution at position 18, with the residue derived from ( S , S )- trans -2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, α-aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the β residue at position 18. Cryoelectron microscopy structures of two GLP-1 analogues bound to the GLP-1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R:Gs complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profiles. Our results advance understanding of the structural underpinnings of receptor activation and introduce tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.

Introduction and Objective: Survodutide (Survo) is a therapy undergoing Phase III studies to investigate its suitability for treating people living with obesity and MASH. Survo is a dual agonist that increases energy expenditure and decreases energy intake via activation of Glucagon Receptor (GCGR) and GLP-1 Receptor (GLP-1R), respectively, leading to up to 19% weight-loss, and 64.5% fibrosis reduction in F2-F3. We investigated the molecular basis of Survo’s dual agonism and how this differs from other GCGR and GLP-1R agonists. Methods: We employed in vitro assays such as cyclic adenosine mono phosphate (cAMP) production to compare Survo to the endogenous peptides GLP-1, glucagon (GCG), and oxyntomodulin (Oxy). Cryogenic electron microscopy (cryo-EM) structures of GCGR and GLP-1R in complex with Survo and G protein were determined at high resolution. Results: Survo is a 29 amino acid (AA) peptide based on GCG with pos 18, 20 and 23 swapped to GLP-1 and pos 16 swapped to exendin-4. Pos 24 and 27-29 were changed to other AA’s and pos 2 to the novel unnatural AA Ac4c. Survo induces cAMP production 4-fold less potent compared to native GLP-1 at GLP-1R (EC50: 20 vs 5 pM, respectively) and 22-times less potent compared to native GCG at GCGR (EC50: 108 vs 5 pM for Survo and GCG, respectively) while resembling the dual agonist Oxy at both receptors (EC50: 36 and 47 pM at GLP-1R and GCGR, respectively). Cryo-EM structures show similar binding poses for Survo compared to other peptide agonists, with differential AA’s showing interactions that explain the assay data. The lysine side chain at pos 24 links to a fatty diacid to improve plasma half-life. Removal of the linker and diacid increased potency at both receptors (EC50: 2 and 23 pM at GLP-1R and GCGR, respectively) with subtle changes in cryo-EM structures. Conclusion: The activation profile of Survo at GCGR and GLP-1R can be rationalized by structural evidence and the gained knowledge will inform therapeutic approaches in both obesity and MASH targeted treatment regimes. We further give insight into the effect of peptide lipidation. E. Yuliantie: None. P.N.H. Trinh: None. F. Bumbak: None. X. Zhang: None. Y. Jiang: None. Q. Ou: None. R. Ebenhoch: Employee; Boehringer-Ingelheim. D. Weichert: Employee; Boehringer-Ingelheim. H. Nar: Employee; Boehringer-Ingelheim. P.M. Sexton: Research Support; AstraZeneca, Pfizer Inc, Boehringer-Ingelheim, Novo Nordisk A/S, Astex Pharmaceuticals. D. Wootten: Research Support; Novo Nordisk A/S, Boehringer-Ingelheim, Pfizer Inc. Stock/Shareholder; Septerna Inc, Dacra Therapeutics. Research Support; AstraZeneca.

The PACAP receptor PAC1 is a Gs-coupled family B1 GPCR for which the highest-affinity endogenous peptide ligands are the pituitary adenylate cyclase-activating peptides PACAP38 and PACAP27, and whose most abundant endogenous ligand is PACAP38. PACAP action at PAC1 is implicated in neuropsychiatric disorders, atherosclerosis, pain chronification, and protection from neurodegeneration and ischemia. As PACAP also interacts with two related receptors, VPAC1 and VPAC2, highly selective ligands, both agonists and antagonists, for PAC1 have been sought. To date, the peptide PACAP(6-38) and polypeptide M65, which is related to maxadilan, a sandfly vasodilator peptide, have been identified as selective for PAC1. Several non-peptide small molecule compounds (SMOLs) have been reported to be specific antagonists at PAC1, albeit there is only limited literature detailing their pharmacology across different systems and within different laboratories. Here, we present a platform of cellular assays for the screening of biologically relevant antagonists at PAC1 and show that some currently proposed SMOL antagonists do not have activity in this cell reporter assay, while we confirm that PACAP(6-38) and M65 are competitive antagonists. We have used this assay system to explore other peptide antagonists at PAC1, guided by molecular dynamics analysis of the PACAP-PAC1 interaction based on cryo-EM structural models of PAC1 complexed with a number of biologically active ligands. The affinity-trap model for the PAC1-ligand interaction successfully predicts the engagement behavior of PACAP27 and PACAP38 peptide-based PAC1 inhibitors. In particular, C-terminal deletants of PACAP(6-38) that maintain equipotency to PACAP(6-38) allow the shorter sequence to function as a scaffold for further peptide-based antagonist exploration.

α- and β-calcitonin gene-related peptides (αCGRP and βCGRP, respectively), together with adrenomedullin (AM) and AM2 are endogenous agonists of the CGRP family of receptors; CGRP receptor (CGRPR), AM1 receptor (AM1R), and AM2 receptor (AM2R). The high sequence homology and similar tissue distribution of αCGRP and βCGRP suggests they have overlapping physiological roles in pain pathways, inflammation, and metabolism, but recent data indicate potential differences in the signaling capabilities of these peptides. However, a comprehensive pharmacological characterization of βCGRP activity, compared to αCGRP, AM, and AM2 across the three CGRP family receptors, is lacking. In this study, we assessed proximal G protein coupling/activation, cognate second messenger production, regulatory protein recruitment and receptor trafficking induced by αCGRP, βCGRP, AM, and AM2 at the CGRPR, AM1R, and AM2R. Our findings revealed a distinct profile of transducer and regulatory protein engagement induced by βCGRP compared to αCGRP across these receptors. The identification of differences in pharmacological profiles for αCGRP and βCGRP indicates that they may have more distinct physiological roles than previously appreciated and may assist in distinguishing the roles of these two peptides for exploitation in targeted drug design.

Membrane cholesterol modulates the ability of cholecystokinin to act at the type 1 cholecystokinin receptor (CCK1R), with elevated levels of this lipid that can be present in obesity, disrupting stimulus-activity coupling. This occurs via direct effect on a cholesterol association (cholesterol recognition/interaction amino acid consensus) motif within CCK1R, whereas an analogous motif is present in the type 2 cholecystokinin receptor (CCK2R) with no functional impact of cholesterol on that receptor. We recently demonstrated that surface residues surrounding this motif in these 2 G protein-coupled receptors could be exchanged as a group to convert their cholesterol sensitivity. We now evaluate each of these residues individually and use molecular dynamics to study cholesterol interactions with this site. These studies show that cholesterol occupies this motif in both CCK1R and CCK2R with similar dominant poses in both receptors in normal, as well as in elevated cholesterol environments. The dominant pose of cholesterol changes in both receptors with elevated cholesterol, shifting proximity from transmembrane segment (TM) 3 toward transmembrane segment 5. Of note, cholesterol residence time at this motif is longer for CCK2R than CCK1R and longer in high cholesterol conditions at both receptors. This suggests that the cholesterol sensitivity of CCK1R is likely a dynamic event mediated by short, transient cholesterol interaction with CCK1R under normal conditions. One residue in position 5.52 was critical for this cholesterol effect on CCK1R, despite the prediction that this is not due to direct interaction with cholesterol. Instead, the packing of hydrophobic residues around 5.52 is tighter in CCK1R than in CCK2R, likely affecting the position and movement of transmembrane segment 5 that are important for stimulus-activity coupling. Significance Statement Membrane cholesterol can affect the function of select G protein-coupled receptors, such as the type 1 cholecystokinin receptor, that are targets for drugs in disease states in which membrane cholesterol can be elevated. This article studied the molecular basis for high cholesterol disruption of stimulus-activity coupling at this receptor using site-directed mutagenesis and molecular dynamics. The findings demonstrate that cholesterol residence time at a key cholesterol association motif within CCK1R is responsible for this effect.

G protein-coupled receptors (GPCRs) are critically important medicinal targets, and the cryogenic electron microscopy (cryo-EM) revolution is providing novel high-resolution GPCR structures at a rapid pace. Orphan G protein-coupled receptors (oGPCRs) are a group of approximately 100 nonolfactory GPCRs for which endogenous ligands are unknown or not validated. The absence of modulating ligands adds difficulties to understanding the physiologic significance of oGPCRs and in the determination of high-resolution structures of isolated receptors that could facilitate drug discovery. Despite the challenges, cryo-EM structures of oGPCR-G protein complexes are emerging. This is being facilitated by numerous developments to stabilize GPCR-G protein complexes such as the use of dominant-negative G proteins, mini-G proteins, complex-stabilizing nanobodies or antibody fragments, and protein tethering methods. Moreover, many oGPCRs are constitutively active, which can facilitate complex formation in the absence of a known activating ligand. Consequently, in addition to providing templates for drug discovery, active oGPCR structures shed light on constitutive GPCR activation mechanisms. These comprise self-activation, whereby mobile extracellular portions of the receptor act as tethered agonists by occupying a canonical orthosteric-binding site in the transmembrane core, constitutive activity due to alterations to conserved molecular switches that stabilize inactive states of GPCRs, as well as receptors activated by cryptic ligands that are copurified with the receptor. Cryo-EM structures of oGPCRs are now being determined at a rapid pace and are expected to be invaluable tools for oGPCR drug discovery. SIGNIFICANCE STATEMENT: Orphan G protein-coupled receptors (GPCRs) provide large untapped potential for development of new medicines. Many of these receptors display constitutive activity, enabling structure determination and insights into observed GPCR constitutive activity including (1) self-activation by mobile receptor extracellular portions that function as tethered agonists, (2) modification of conserved motifs canonically involved in receptor quiescence and/or activation, and (3) activation by cryptic lipid ligands. Collectively, these studies advance fundamental understanding of GPCR function and provide opportunities for novel drug discovery.

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight.

The pituitary adenylate cyclase-activating polypeptide (PACAP) 1 receptor (PAC1R) is a class B1 G protein–coupled receptor activated by the endogenous peptide agonists PACAP and vasoactive intestinal peptide (VIP). Alternate splicing within the receptor extracellular domain (ECD) generates the PAC1R short variant (PAC1sR) that has selectively enhanced VIP function compared to the full-length, PAC1R null variant (PAC1nR). However, to date, a comprehensive pharmacological assessment of the downstream signaling outcomes of PAC1sR activation compared to PAC1nR has not been performed, and little information is available to mechanistically understand how ECD splicing may alter ligand engagement. Here, we demonstrated that VIP, but not PACAP, has globally enhanced activity across a broad range of functional endpoints at PAC1sR compared to PAC1nR. Cryo-EM structures of VIP-bound, stimulatory G protein (G s )-coupled PAC1sR and PAC1nR, supported by molecular dynamics (MD) simulations, demonstrate transient engagement of the null loop in PAC1nR, which is absent in PAC1sR, with residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD. These interactions result in differential engagement of VIP with these domains and the top of TM2/ECL1 with PAC1sR and PAC1nR. Moreover, MD simulations predicted differential interactions of the G s protein with the two PAC1R variants when bound by VIP that correlate with a greater allosteric influence of the G s protein on VIP affinity at the PAC1sR, relative to PAC1nR. Our study provides insights into the structural basis and functional consequences of PAC1R ECD splicing, increasing understanding of PAC1R ligand selectivity and signaling.

Efficient and precise delivery of mRNA is critical to advance mRNA therapies beyond their current use as vaccines. Lipid nanoparticles (LNPs) efficiently encapsulate and protect mRNA, but non-specific cellular uptake may lead to off-target delivery and minimal delivery to target cells. Functionalizing LNPs with antibodies enables targeted mRNA delivery, but traditional modification techniques require complex conjugation and purification, which often reduces antibody affinity. Here we present a simple method for capturing antibodies in their optimal orientation on LNPs, without antibody modification or complex purification. This strategy uses an optimally oriented anti-Fc nanobody on the LNP surface to capture antibodies, resulting in protein expression levels more than 1,000 times higher than non-targeted LNPs and more than 8 times higher than conventional antibody functionalization techniques. These precisely targeted LNPs showed highly efficient in vivo targeting to T cells, with minimal delivery to other immune cells. This approach enables the rapid development of targeted LNPs and has the potential to broaden the use of mRNA therapies.