G Protein-coupled Receptors Ligands Research Articles

Alterations in mouse visceral adipose tissue mRNA expression of islet G-protein-coupled receptor ligands in obesity.

Adipose tissue mass expansion in obesity leads to alterations in expression and secretion of adipokines, some of which may alter islet function by binding to G-protein-coupled receptors (GPCRs) expressed by islets. We have therefore quantified expression of mRNAs encoding islet GPCR ligands in visceral adipose tissue retrieved from lean and diet-induced obese mice to determine alterations in islet GPCR ligand mRNAs in obesity. Epididymal adipose tissue was retrieved from C57BL/6 mice that had been maintained on a control-fat diet (10% fat) or high-fat diet (60% fat) for 16 weeks and RT-qPCR was used to quantify mRNAs encoding ligands for islet GPCRs. Of the 155 genes that encode ligands for islet GPCRs, 45 and 40 were expressed in visceral adipose tissue retrieved from lean and obese mice respectively. The remaining mRNAs were either expressed at trace level (0.0001% to 0.001% relative to Actb expression) or absent (<0.0001%). Obesity was associated with significant alterations in GPCR ligand mRNA expression in visceral adipose tissue, some of which encode for peptides with established effects on islet function (e.g. neuropeptide Y), or for GPCR ligands that have not previously been investigated for their effects on islets (e.g. (C-C motif) ligand 4; Ccl4). Mouse visceral adipose tissue showed significant alterations in expression of mRNAs encoding islet GPCR ligands in obesity. Our data point to ligands of interest for future research on adipose-islet crosstalk via secreted ligands acting at islet GPCRs. Such research may identify islet GPCRs with therapeutic potential for T2D.

Differential Effects of Beta-Hydroxybutyrate Enantiomers on Induced Pluripotent Stem Derived Cardiac Myocyte Electrophysiology.

Beta-hydroxybutyrate (βOHB), along with acetoacetate and acetone, are liver-produced ketone bodies that are increased after fasting or prolonged exercise as an alternative fuel source to glucose. βOHB, as the main circulating ketone body, is not only a G-protein coupled receptor ligand but also a histone deacetylases inhibitor, prompting the reexamination of its role in health and disease. In this study, we compared the effects of two commercial βOHB formulations an enantiomer R βOHB and a racemic mixture ±βOHB on induced pluripotent stem cell cardiac myocytes (iPS-CMs) electrophysiology. Cardiac myocytes were cultured in R βOHB or ±βOHB for at least ten days after lactate selection. Flouvolt or Fluo-4 was used to assay iPS-CMs electrophysiology. We found that while both formulations increased the optical potential amplitude, R βOHB prolonged the action potential duration but ±βOHB shortened the action potential duration. Moreover, ±βOHB increased the peak calcium transient but R βOHB reduced the peak calcium transient. Co-culturing with glucose or fatty acids did not ameliorate the effects, suggesting that βOHB was more than a fuel source. The effect of βOHB on iPS-CMs electrophysiology is most likely stereoselective, and care must be taken to evaluate the role of exogenous βOHB in health and disease.

Left ventricular assist device bioinformatics identify possible hubgenes and regulatory networks involved in the myocardium of patients with left ventricular assist device.

ObjectiveThe aim of this study was to clarify the changes of myocardial gene expression profile after left ventricular assist device (LVAD) implantation and the related molecular biological significance.MethodsA thorough bioinformatic analysis to evaluate the changes in gene expression profile in patients pre-LVAD and post-LVAD was conducted. Four relevant gene expression datasets—GSE430, GSE974, GSE21610, and GSE52601 from Gene Expression Omnibus (GEO) database were downloaded. Analysis of GEO2R, Gene Ontology (GO), protein-protein interaction (PPI) were used to determine differentially expressed genes (DEGs) and their function, respectively.ResultsA total of 37 DEGs were identified, including 26 down-regulated and 11 up-regulated genes. The molecular function of DEGs were enriched in “cytokine activity,” “neurotransmitter binding,” “receptor ligand activity.” The gene set enrichment analysis (GSEA) revealed an overall marked increase of neutrophil degranulation signaling, closely correlated with the G protein coupled receptor (GPCR)—ligand binding process after LVAD assistance. 16 hubgenes in these DEGs were further selected and the biological process involved is mainly related to positive regulation of leukocyte chemotaxis mediated by chemokines.ConclusionInflammatory signaling pathway is crucial for the pathophysiology after LVAD implantation. Chemokines mediate cardiac inflammatory response and tissue remodeling after LVAD implantation through GPCR—ligand binding.

Genetically encoded fluorescent biosensors for GPCR research.

G protein-coupled receptors (GPCRs) regulate a wide range of physiological and pathophysiological cellular processes, thus it is important to understand how GPCRs are activated and function in various cellular contexts. In particular, the activation process of GPCRs is dynamically regulated upon various extracellular stimuli, and emerging evidence suggests the subcellular functions of GPCRs at endosomes and other organelles. Therefore, precise monitoring of the GPCR activation process with high spatiotemporal resolution is required to investigate the underlying molecular mechanisms of GPCR functions. In this review, we will introduce genetically encoded fluorescent biosensors that can precisely monitor the real-time GPCR activation process in live cells. The process includes the binding of extracellular GPCR ligands, conformational change of GPCR, recruitment of G proteins or β-arrestin, GPCR internalization and trafficking, and the GPCR-related downstream signaling events. We will introduce fluorescent GPCR biosensors based on a variety of strategies such as fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), circular permuted fluorescent protein (cpFP), and nanobody. We will discuss the pros and cons of these GPCR biosensors as well as their applications in GPCR research.

Pharmacological targeting of G protein-coupled receptor heteromers

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4–116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4–116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson’s disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, and inverse agonists) of G-protein coupled receptor ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available nonradioligand ready-to-use kits and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

G protein-coupled receptors (GPCRs) regulate processes ranging from immune responses to neuronal signaling. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additionally, dissecting cell type-specific responses is challenging when the same GPCR is expressed on different cells within a tissue. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that bind clozapine-N-oxide and mimic a GPCR-of-interest. We show that chimeric DREADD-β2AR triggers responses comparable to β2AR on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate β2AR-mediated filopodia formation in microglia, an immune cell capable of driving central nervous system inflammation. When dissecting microglial inflammation, we included two additional DREADD-based chimeras mimicking microglia-enriched GPR65 and GPR109A. DREADD-β2AR and DREADD-GPR65 modulate the inflammatory response with high similarity to endogenous β2AR, while DREADD-GPR109A shows no impact. Our DREADD-based approach allows investigation of cell type-dependent pathways without known endogenous ligands.

Finding the Perfect Fit: Conformational Biosensors to Determine the Efficacy of GPCR Ligands.

G protein-coupled receptors (GPCRs) are highly druggable targets that adopt numerous conformations. A ligand's ability to stabilize specific conformation(s) of its cognate receptor determines its efficacy or ability to produce a biological response. Identifying ligands that produce different receptor conformations and potentially discrete pharmacological effects (e.g., biased agonists, partial agonists, antagonists, allosteric modulators) is a major goal in drug discovery and necessary to develop drugs with better effectiveness and fewer side effects. Fortunately, direct measurements of ligand efficacy, via receptor conformational changes are possible with the recent development of conformational biosensors. In this review, we discuss classical efficacy models, including the two-state model, the ternary-complex model, and multistate models. We describe how nanobody-, transducer-, and receptor-based conformational biosensors detect and/or stabilize specific GPCR conformations to identify ligands with different levels of efficacy. In particular, conformational biosensors provide the potential to identify and/or characterize therapeutically desirable but often difficult to measure conformations of receptors faster and better than current methods. For drug discovery/development, several recent proof-of-principle studies have optimized conformational biosensors for high-throughput screening (HTS) platforms. However, their widespread use is limited by the fact that few sensors are reliably capable of detecting low-frequency conformations and technically demanding assay conditions. Nonetheless, conformational biosensors do help identify desirable ligands such as allosteric modulators, biased ligands, or partial agonists in a single assay, representing a distinct advantage over classical methods.

Multifunctional role of GPCR signaling in epithelial tube formation

Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G-protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the medioapical region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data support a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors.

The two human melatonin receptors MT1 and MT2, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [3H]melatonin and 2-[125I]iodomelatonin (2-[125I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time. However, developing such assays for the two melatonin receptors depends on the availability of fluorescent tracers, which has been challenging predominantly owing to their narrow ligand entry channel and small ligand binding pocket. Here, we generated a set of melatonergic fluorescent tracers and used NanoBRET to evaluate their engagement with MT1 and MT2 receptors that are genetically fused to an N-terminal luminogenic HiBiT-peptide. We identified several nonselective and subtype-selective tracers. Among the selective tracers, PBI-8238 exhibited high nanomolar affinity to MT1, and PBI-8192 exhibited low nanomolar affinity to MT2. The pharmacological profiles of both tracers were in good agreement with those obtained with the current standard 2-[125I]-MLT radioligand. Molecular docking and mutagenesis studies suggested the binding mode of PBI-8192 in MT2 and its selectivity over MT1. In conclusion, we describe the development of the first nonradioactive, real-time binding assays for melatonin receptors expressed at the cell surface of living cells that are likely to accelerate drug discovery for melatonin receptors.

Identification of A2BAR as a potential target in colorectal cancer using novel fluorescent GPCR ligands

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.

Recent progress in assays for GPCR drug discovery.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors, are the largest family of cell surface receptors in eukaryotes. There are ∼800 GPCRs in human, regulating diverse physiological processes. The GPCRs are the most intensively studied drug targets. Drugs that target GPCRs account for about a quarter of the global market share of therapeutic drugs. Therefore, to develop physiologically relevant and robust assays to search new GPCR ligands or modulators remain the major focus of drug discovery research worldwide. Early functional GPCR assays mainly depend on the measurement of G-protein-mediated second messenger generation. Recent developments in GPCR biology indicate that the signaling of these receptors is much more complex than the oversimplified classical view. The GPCRs have been found to activate multiple G proteins simultaneously and induce β-arrestin-mediated signaling. They have also been found to interact with other cytosolic scaffolding proteins and form dimer or heteromer with GPCRs or other transmembrane proteins. Here, we mainly discuss technologies focused on detecting protein-protein interactions, such as fluorescence resonance energy transfer/bioluminescence resonance energy transfer (FRET/BRET), NanoLuc binary technology (NanoBiT), Tango, etc., and their applications in measuring GPCRs interacting with various signaling partners. In the final part, we also discuss the species differences in GPCRs when using animal models to study the in vivo functions of GPCR ligands, and possible ways to solve this problem with modern genetic tools.

What Makes GPCRs from Different Families Bind to the Same Ligand?

G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptor proteins with important functions in signal transduction and often serve as therapeutic drug targets. With the rapidly growing public data on three dimensional (3D) structures of GPCRs and GPCR-ligand interactions, computational prediction of GPCR ligand binding becomes a convincing option to high throughput screening and other experimental approaches during the beginning phases of ligand discovery. In this work, we set out to computationally uncover and understand the binding of a single ligand to GPCRs from several different families. Three-dimensional structural comparisons of the GPCRs that bind to the same ligand revealed local 3D structural similarities and often these regions overlap with locations of binding pockets. These pockets were found to be similar (based on backbone geometry and side-chain orientation using APoc), and they correlate positively with electrostatic properties of the pockets. Moreover, the more similar the pockets, the more likely a ligand binding to the pockets will interact with similar residues, have similar conformations, and produce similar binding affinities across the pockets. These findings can be exploited to improve protein function inference, drug repurposing and drug toxicity prediction, and accelerate the development of new drugs.

Loss of Biased Signaling Specificity of the Angiotensin II Type 1 Receptor in Overexpressed Systems

G protein‐coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β‐arrestins. [Sarcosine1Ile4Ile8]‐angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in multiple primary cell lines. Despite this, SII induces phosphorylation of the extracellular signal‐regulated kinases 1 and 2 (ERK1/2) through β‐arrestin‐2‐dependent mechanisms. However, studies reliant on exogenous overexpression of the AT1R indicate that SII is a partial agonist for G protein signaling and lacks β‐arrestin‐exclusive signaling specificity. We investigated this apparent discrepancy by profiling changes in signaling specificity at the AT­­1R with increasing levels of receptor expression. We hypothesized that overexpression of the AT1R causes loss of signaling pathway specificity, such that ligands capable of inducing only β‐arrestin‐mediated responses at lower physiologic receptor expression levels gain the ability to activate G proteins upon receptor overexpression. We established a tetracycline‐inducible cellular system for titratable expression of the AT1R to assess signaling responses at varying levels of receptor expression when stimulated with AngII, SII, and four other AngII analogs with different signaling biases. Unbiased and G protein‐biased ligands activated dose‐dependent calcium responses at all levels of AT1R expression. In contrast, β‐arrestin‐biased ligands induced dose‐dependent calcium signaling only at higher levels of receptor overexpression. Using inhibitors of different G proteins, we demonstrated that both Gi and Gq mediated this overexpression‐dependent calcium signaling by β‐arrestin‐biased ligands. All ligands induced ERK1/2 phosphorylation at receptor levels below those required for calcium signaling by β‐arrestin‐biased ligands. Thus, receptor overexpression causes loss of biased signaling specificity of AT1R ligands, highlighting the potential risks of using such systems to simulate GPCR ligand behavior in physiologically relevant contexts.

High‐throughput CRISPR for Generating Therapeutic Nanobodies

Engineered antibodies have had a widespread impact throughout biomedical sciences, from their use as fundamental research tools to their employment as therapeutics. Closely related nanobodies, derived from heavy‐chain only antibodies, have begun gaining much attention as alternatives with improved pharmacological properties. Containing only the antigen recognition components, nanobodies maintain the exceptional specificity of conventional antibodies but their simplistic design leads to increased stability and decreased toxicity. In recent years, nanobodies have proven especially useful for studying G protein‐coupled receptors (GPCRs), a class of membrane receptors responsible for activating intracellular proteins (e.g., Gα proteins) and initiating subsequent signaling pathways. Most GPCR‐targeting nanobodies to date have been designed as crystallographic chaperones for stabilizing the GPCR‐Gα interaction for in‐depth structural insights. However, harnessing the intrinsic specificity provided by nanobodies for therapeutic purposes has been limited to few receptors. In this study, we present a unique platform for discovering functionally selective nanobodies for a broad range of GPCRs. Using a yeast model proven invaluable for discovering GPCR ligands and structure‐stabilizing nanobodies, we engineered a system for rapidly identifying nanobody‐based therapeutic candidates. In this system, each cell uniquely contains 1) a tethered nanobody displayed outside the cell membrane, 2) a human GPCR, 3) one of ten Gα proteins for characterizing GPCR‐Gα interactions, and 4) a fluorescent reporter induced upon GPCR/Gα activation. By combining all components into a single cell, we can directly access the therapeutic potential of each nanobody‐GPCR combination by monitoring the (in)activity of relevant signaling pathways inspired from this interaction. To date, we have completed the initial engineering stages of this platform and showcased its utility through pharmacological activation of various GPCRs (e.g., SSTR5, AGTR1, CXCR4, and C5aR) via tethered peptide/protein agonists. Next, we will use high‐throughput CRISPR to generate an extensive nanobody library for rapidly identifying and functionally quantifying GPCR‐targeting nanobodies. Using this approach for a large variety of GPCRs, this platform will provide a vast collection of nanobody‐based therapeutic candidates, and with minimal modifications may be expanded for identifying therapeutics for other classes of membrane receptors.

Single-cell imaging of ERK and Akt activation dynamics and heterogeneity induced by G-protein-coupled receptors.

ABSTRACTKinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters. The kinases were activated with GPCR ligands. We observed ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

Targeting GPCRs and Their Signaling as a Therapeutic Option in Melanoma.

Simple SummarySixteen G-protein-coupled receptors (GPCRs) have been involved in melanogenesis or melanomagenesis. Here, we review these GPCRs, their associated signaling, and therapies.G-protein-coupled receptors (GPCRs) serve prominent roles in melanocyte lineage physiology, with an impact at all stages of development, as well as on mature melanocyte functions. GPCR ligands are present in the skin and regulate melanocyte homeostasis, including pigmentation. The role of GPCRs in the regulation of pigmentation and, consequently, protection against external aggression, such as ultraviolet radiation, has long been established. However, evidence of new functions of GPCRs directly in melanomagenesis has been highlighted in recent years. GPCRs are coupled, through their intracellular domains, to heterotrimeric G-proteins, which induce cellular signaling through various pathways. Such signaling modulates numerous essential cellular processes that occur during melanomagenesis, including proliferation and migration. GPCR-associated signaling in melanoma can be activated by the binding of paracrine factors to their receptors or directly by activating mutations. In this review, we present melanoma-associated alterations of GPCRs and their downstream signaling and discuss the various preclinical models used to evaluate new therapeutic approaches against GPCR activity in melanoma. Recent striking advances in our understanding of the structure, function, and regulation of GPCRs will undoubtedly broaden melanoma treatment options in the future.

Multi-source transfer learning with Graph Neural Network for excellent modelling the bioactivities of ligands targeting orphan G protein-coupled receptors.

G protein-coupled receptors (GPCRs) have been the targets for more than 40% of the currently approved drugs. Although neural networks can effectively improve the accuracy of prediction with the biological activity, the result is undesirable in the limited orphan GPCRs (oGPCRs) datasets. To this end, we proposed Multi-source Transfer Learning with Graph Neural Network, called MSTL-GNN, to bridge this gap. Firstly, there are three ideal sources of data for transfer learning, oGPCRs, experimentally validated GPCRs, and invalidated GPCRs similar to the former one. Secondly, the SIMLEs format GPCRs convert to graphics, and they can be the input of Graph Neural Network (GNN) and ensemble learning for improving prediction accuracy. Finally, our experiments show that MSTL-GNN remarkably improves the prediction of GPCRs ligand activity value compared with previous studies. On average, the two evaluation indexes we adopted, R2 and Root-mean-square deviation (RMSE). Compared with the state-of-the-art work MSTL-GNN increased up to 67.13% and 17.22%, respectively. The effectiveness of MSTL-GNN in the field of GPCR Drug discovery with limited data also paves the way for other similar application scenarios.

Endogenous Peptide Inhibitors of HIV Entry.

The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1β specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.

GPR15–C10ORF99 functional pairing initiates colonic Treg homing in amniotes

Regulatory T lymphocyte (Treg) homing reactions mediated by G protein‐coupled receptor (GPCR)–ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15–C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short‐chain fatty acid‐C10ORF99/GPR15‐induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15–C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR–ligand pairing leads to physiological adaptation during vertebrate evolution.