Absence of Functional Autoantibodies Targeting Angiotensin II Type 1 Receptor (ATR) and Endothelin-1 Type A Receptor (ETR) in Circulation and Purified IgG from Patients with Systemic Sclerosis.

Objective: Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. While previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II type 1 receptor (ATR) and endothelin-1 type A receptor (ETR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays.

GPCR Biosensors to Study Conformational Dynamics and Signaling in Drug Discovery.

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane signal transducers that facilitate the flow of chemical signals across membranes. GPCRs are a desirable class of drug targets, and the activation and deactivation dynamics of these receptors are widely studied. Multidisciplinary approaches for studying GPCRs, such as downstream biochemical signaling assays, cryo-electron microscopy structural determinations, and molecular dynamics simulations, have provided insights concerning conformational dynamics and signaling mechanisms.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 β 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.

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.

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

Frizzleds (FZD) are G protein–coupled 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.

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.

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.

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.

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.

Combined Inhibition of Gα 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 (Gα) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gα have shown promising preclinical results, but their therapeutic activity in distinct Gα mutational contexts and have remained elusive.