Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

Application of bioluminescence resonance energy transfer to quantitate cell-surface expression of membrane proteins.

We report a bioluminescence resonance energy transfer (BRET) assay to quantitate the fraction of an engineered membrane protein at the cell surface versus inside the cell. As test cases, we engineered two different G protein-coupled receptors (GPCRs) in which a NanoLuc luciferase (NLuc) and a HaloTag are fused to the extracellular amino-terminal tail of the receptors. We then employed a pulse-chase labeling approach relying on two different fluorescent dyes with distinctive cell permeability properties. The dyes are efficiently excited by luminescence from NLuc, but are spectrally distinct. Measuring BRET from the chemiluminescence of the NLuc to the fluorophores bound to the HaloTag minimizes the limitations of in-cell fluorescence resonance energy transfer (FRET)-based approaches such as photobleaching and autofluorescence. The BRET surface expression assay can quantitatively differentiate between the labeling of receptors at the cell surface and receptors inside of the cell. The assay is shown to be quantitative and robust compared with other approaches to measure cell surface expression of membrane proteins such as enzyme-linked immunosorbent assay or immunoblotting, and significantly increases the throughput because the assay is designed to be carried out in microtiter plate format.

Expanding the GPCR-RAMP interactome.

Receptor activity-modifying proteins (RAMPs) can form complexes with G protein-coupled receptors (GPCRs) and regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a complete interactome between GPCRs and the three RAMPs, we developed a customized library of 215 Dual Epitope-Tagged (DuET) GPCRs representing all GPCR subfamilies. Using a multiplexed suspension bead array (SBA) assay, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for native interactions in three cell lines and found 23 GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective GPCR-targeted therapeutics.

Multiplexed selectivity screening of anti-GPCR antibodies.

G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR subfamilies. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound off-target, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs and for detecting pathological auto-Abs against GPCRs.

Itch receptor MRGPRX4 interacts with the receptor activity-modifying proteins.

Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.

Bioorthogonal Tethering Enhances Drug Fragment Affinity for G Protein-Coupled Receptors in Live Cells.

G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C-C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels-Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.

Autoantibodies targeting G protein-coupled receptors: An evolving history in autoimmunity. Report of the 4th international symposium.

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions.

Interdisciplinary biophysical studies of membrane proteins bacteriorhodopsin and rhodopsin.

The centenary of the birth of H. Gobind Khorana provides an auspicious opportunity to review the origins and evolution of parallel advances in biophysical methodology and molecular genetics technology used to study membrane proteins. Interdisciplinary work in the Khorana laboratory in the late 1970s and for the next three decades led to productive collaborations and fostered three subsequent scientific generations whose biophysical work on membrane proteins has led to detailed elucidation of the molecular mechanisms of energy transduction by the light-driven proton pump bacteriorhodopsin (bR) and signal transduction by the G protein-coupled receptor (GPCR) rhodopsin. This review will highlight the origins and advances of biophysical studies of membrane proteins made possible by the application of molecular genetics approaches to engineer site-specific alterations of membrane protein structures.

Elucidating the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins.

G protein-coupled receptors (GPCRs) are known to interact with several other classes of integral membrane proteins that modulate their biology and pharmacology. However, the extent of these interactions and the mechanisms of their effects are not well understood. For example, one class of GPCR-interacting proteins, receptor activity-modifying proteins (RAMPs), comprise three related and ubiquitously expressed single-transmembrane span proteins. The RAMP family was discovered more than two decades ago, and since then GPCR-RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several secretin (class B) GPCRs, most notably the calcitonin receptor-like receptor. Recent bioinformatics and multiplexed experimental studies suggest that GPCR-RAMP interactions might be much more widespread than previously anticipated. Recently, cryo-electron microscopy has provided high-resolution structures of GPCR-RAMP-ligand complexes, and drugs have been developed that target GPCR-RAMP complexes. In this review, we provide a summary of recent advances in techniques that allow the discovery of GPCR-RAMP interactions and their functional consequences and highlight prospects for future advances. We also provide an up-to-date list of reported GPCR-RAMP interactions based on a review of the current literature. SIGNIFICANCE STATEMENT: Receptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR)biology and pharmacology. The application of new methodologies to study membrane protein-protein interactions suggests that RAMPs interact with many more GPCRs than had been previously known. These findings, especially when combined with structural studies of membrane protein complexes, have significant implications for advancing GPCR-targeted drug discovery and the understanding of GPCR pharmacology, biology, and regulation.

Genetic code expansion to enable site-specific bioorthogonal labeling of functional G protein-coupled receptors in live cells.

For use in site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells, we developed a luciferase-based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side-by-side the efficiency of incorporation of three different noncanonical amino acids [4-azido-l-phenylalanine (azF), cyclopropene-l-lysine (CpK), and trans-cyclooct-2-en-l-lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope-tagged C-C chemokine receptor 5 (CCR5)-RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell-based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine-containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine-containing compounds using the inverse electron demand Diels-Alder ligation was overall slightly more efficient than other reactions tested.

The Coronavirus Calendar (CoronaCal): a simplified SARS-CoV-2 test system for sampling and retrospective analysis.

Objectives: To develop a biological diary (CoronaCal) that allows anyone in the community to collect and store serial saliva samples and chart symptoms on ordinary printer paper. Methods: Diaries were analyzed for the presence of SARS-CoV-2 RNA using established polymerase chain reaction (PCR) procedures. CoronaCal diaries were distributed to volunteer subjects in the community during the peak of the COVID-19 outbreak in New York. Volunteers collected their own daily saliva samples and self-reported symptoms. Results: SARS-CoV-2 RNA extracted from CoronaCals was measured using qPCR and RNA levels were correlated with reported symptoms. SARS-CoV-2 RNA was detected in CoronaCals from nine of nine people with COVID-19 symptoms or exposure to someone with COVID-19, and not in one asymptomatic person. CoronaCals were stored for up to 70 days at room temperature during collection and then frozen for up to four months before analysis, suggesting that SARS-CoV-2 RNA is stable once dried onto paper. Conclusions: Sampling saliva on simple paper provides a useful method to study the natural history and epidemiology of COVID-19. The CoronaCal collection and testing method is easy to implement, inexpensive, non-invasive and scalable. The approach can inform the historical and epidemiological understanding of infections in individuals and populations.

Human Islet Amyloid Polypeptide (hIAPP) Protofibril-Specific Antibodies for Detection and Treatment of Type 2 Diabetes.

Type 2 diabetes mellitus (T2D) is a major public health concern and is characterized by sustained hyperglycemia due to insulin resistance and destruction of insulin-producing ß cells. One pathological hallmark of T2D is the toxic accumulation of human islet amyloid polypeptide (hIAPP) aggregates. Monomeric hIAPP is a hormone normally co-secreted with insulin. However, increased levels of hIAPP in prediabetic and diabetic patients can lead to the formation of hIAPP protofibrils, which are toxic to ß cells. Current therapies fail to address hIAPP aggregation and current screening modalities do not detect it. Using a stabilizing capping protein, monoclonal antibodies (mAbs) can be developed against a previously nonisolatable form of hIAPP protofibrils, which are protofibril specific and do not engage monomeric hIAPP. Shown here are two candidate mAbs that can detect hIAPP protofibrils in serum and hIAPP deposits in pancreatic islets in a mouse model of rapidly progressing T2D. Treatment of diabetic mice with the mAbs delays disease progression and dramatically increases overall survival. These results demonstrate the potential for using novel hIAPP protofibril-specific mAbs as a diagnostic screening tool for early detection of T2D, as well as therapeutically to preserve ß cell function and target one of the underlying pathological mechanisms of T2D.

Getting to the heart of cannabis health risks.

In this issue of Cell, Wei et al. show that the increased cardiovascular risks associated with cannabis use are mediated by proinflammatory cannabinoid 1 (CB1) receptor signaling, which can be ameliorated with the natural antioxidant agent genistein.

FRET sensors reveal the retinal entry pathway in the G protein-coupled receptor rhodopsin.

The photoreceptor rhodopsin (Rho) becomes active when a tethered inverse agonist ligand (11CR) is photoconverted to an agonist (ATR). The ligand-binding pocket of inactive rhodopsin is completely enclosed, whereas active rhodopsin displays pores accessible from the lipid bilayer. Stabilization of active rhodopsin impedes 11CR binding and photoreceptor dark adaptation. Here, we used genetic code expansion and bioorthogonal labeling to engineer Rho mutants that serve as FRET sensors for measuring 11CR binding kinetics and energetics. We found that mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics but not agonist release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings provide a conceptual framework for understanding the function of G protein-coupled receptors with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.

Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

Frizzleds (FZD1­10) are G protein­coupled receptors containing an extracellular cysteine-rich domain (CRD) binding Wingless/Int-1 lipoglycoproteins (WNTs). Despite the role of WNT/FZD signaling in health and disease, our understanding of how WNT binding is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics. To clarify how WNT binding to FZD translates into receptor dynamics, we devised conformational FZD-CRD biosensors based on bioluminescence resonance energy transfer (BRET). Using FZD with N-terminal nanoluciferase (Nluc) and fluorescently labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct signaling pathways and that CRD dynamics are not required for WNT/ß-catenin signaling. Thus, these FZD-CRD biosensors provide insights into binding, activation, and signaling processes in FZDs. The sensor design is broadly applicable to explore ligand-induced dynamics also in other membrane receptors.

Archiving time series sewage samples as biological records of built environments.

This commentary encourages the regular archiving of nucleic-acid-stabilized serial samples of wastewaters and/or sewage. Stabilized samples would facilitate retrospective reconstitution of built environments' biological fluids. Biological time capsules would allow retrospective searches for nucleic acids from viruses such as SARS-CoV-2. Current resources for testing need not be diverted if samples are saved in case they become important in the future. Systematic storage would facilitate investigation into the origin and prevalence of viruses and other agents. Comparison of prevalence data from individual and clinical samplings with community wastewater would allow valuable comparison, contrast and correlation among different testing modalities. Current interest is focused on SARS-CoV-2, but archived samples could become valuable in many contexts including surveys for other infectious and chemical agents whose identity is not currently known. Archived time series of wastewater will take their place alongside other biological repositories and records including those from medical facilities, museums, eDNA, living cell and tissue collections. Together these will prove invaluable records of the evolving Anthropocene.

DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Principles and practice for SARS-CoV-2 decontamination of N95 masks with UV-C.

A mainstay of personal protective equipment during the coronavirus disease 2019 pandemic is the N95 filtering facepiece respirator. N95 respirators are commonly used to protect healthcare workers from respiratory pathogens, including the novel coronavirus severe acute respiratory syndrome coronavirus 2, and are increasingly employed by other frontline workers and the general public. Under routine circumstances, these masks are disposable, single-use items, but extended use and reuse practices have been broadly enacted to alleviate critical supply shortages during the coronavirus disease 2019 pandemic. Although extended-time single use presents a low risk of pathogen transfer, repeated donning and doffing of potentially contaminated masks presents increased risk of pathogen transfer. Therefore, efficient and safe decontamination methods for N95 masks are needed to reduce the risk of reuse and mitigate local supply shortages. Here, we review the available literature concerning use of germicidal ultraviolet-C (UV-C) light to decontaminate N95 masks. We propose a practical method for repeated point-of-use decontamination using commercially available UV-C cross-linker boxes from molecular biology laboratories to expose each side of the mask to 800-1200 mJ/cm2 of UV-C. We measure the dose that penetrated to the interior of the respirators and model the potential germicidal action on coronaviruses. Our experimental results, in combination with modeled data, suggest that such a UV-C treatment cycle should induce a >3-log-order reduction in viral bioburden on the surface of the respirators and a 2-log-order reduction throughout the interior. We find that a dose 50-fold greater does not impair filtration or fit of 3M 8210 N95 masks, indicating that decontamination can be performed repeatedly. As such, UV-C germicidal irradiation is a practical strategy for small-scale point-of-use decontamination of N95s.

Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling.

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.

Combined Inhibition of Gαq and MEK Enhances Therapeutic Efficacy in Uveal Melanoma.

PURPOSE: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (Gαq) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gαq have shown promising preclinical results, but their therapeutic activity in distinct Gαq mutational contexts and in vivo have remained elusive. EXPERIMENTAL DESIGN: We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of Gαq inhibition as a single agent and in combination with MEK inhibition. RESULTS: We demonstrate that the Gαq inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. CONCLUSIONS: These data suggest that the combination of Gαq and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting Gαq in uveal melanoma.See related commentary by Neelature Sriramareddy and Smalley, p. 1217.