Highly multiplexed bioactivity screening reveals human and microbiota metabolome-GPCRome interactions.

The human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.

Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

Structural insights into membrane adenylyl cyclases, initiators of cAMP signaling.

Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. As effector proteins of G protein-coupled receptors and other signaling pathways, ACs receive and amplify signals from the cell surface, translating them into biochemical reactions in the intracellular space and integrating different signaling pathways. Despite their importance in signal transduction and physiology, our knowledge about the structure, function, regulation, and molecular interactions of ACs remains relatively scarce. In this review, we summarize recent advances in our understanding of these membrane enzymes.

Structural view of G protein-coupled receptor signaling in the retinal rod outer segment.

Visual phototransduction is the most extensively studied G protein-coupled receptor (GPCR) signaling pathway because of its quantifiable stimulus, non-redundancy of genes, and immense importance in vision. We summarize recent discoveries that have advanced our understanding of rod outer segment (ROS) morphology and the pathological basis of retinal diseases. We have combined recently published cryo-electron tomography (cryo-ET) data on the ROS with structural knowledge on individual proteins to define the precise spatial limitations under which phototransduction occurs. Although hypothetical, the reconstruction of the rod phototransduction system highlights the potential roles of phosphodiesterase 6 (PDE6) and guanylate cyclases (GCs) in maintaining the spacing between ROS discs, suggesting a plausible mechanism by which intrinsic optical signals are generated in the retina.

Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV.

CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.

Two C-terminal isoforms of Aplysia tachykinin-related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Molecular determinants of neuropeptide-mediated activation mechanisms in tachykinin NK1 and NK2 receptors.

Substance P and neurokinin A are closely related neuropeptides belonging to the tachykinin family. Their receptors are neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R), G protein-coupled receptors that transmit Gs and Gq-mediated downstream signaling. We investigate the importance of sequence differences at the bottom of the receptor orthosteric site for activity and selectivity, focusing on residues that closely interact with the C-terminal methionine of the peptide ligands. We identify a conserved serine (NK1R-S2977.45) and the position of the tryptophan residue within the canonical "toggle switch" motif, CWxP of TM6, neighboring a phenylalanine in NK1R (NK1R-F2646.51) and a tyrosine in NK2R (NK2R-Y2666.51), giving rise to distinct micro-environments for the neuropeptide C-terminals. Mutating these residues results in dramatic activity changes in both NK1R and NK2R due to a close interaction between the ligand and toggle switch. Structural analysis of active and inactive NKR structures suggest only a minor change in sidechain rotation of toggle switch residues upon activation. However, extensive molecular dynamics simulations of receptor:neuropeptide:G protein complexes indicate that a major, concerted motion happens in the toggle switch tryptophan indole group and the sidechains of the micro-switch motif PIF. This rotation establishes a tight hydrogen bond interaction from the tryptophan indole to the conserved serine (NK1R-S2977.45) and a mainchain carbonyl (NK1R-A2947.41) in the kink of TM7. This interaction facilitates communication with the NPxxY micro-switch motif of TM7, resulting in stabilization of the G protein binding region. NK1R-S2977.45 is consequently identified as a central hub for the activation of NKRs.

Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation.

Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.

Interaction networks within disease-associated GαS variants characterized by an integrative biophysical approach.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

From membrane to nucleus: A three-wave hypothesis of cAMP signaling.

For many decades, our understanding of G protein-coupled receptor (GPCR) activity and cyclic AMP (cAMP) signaling was limited exclusively to the plasma membrane. However, a growing body of evidence has challenged this view by introducing the concept of endocytosis-dependent GPCR signaling. This emerging paradigm emphasizes not only the sustained production of cAMP but also its precise subcellular localization, thus transforming our understanding of the spatiotemporal organization of this process. Starting from this alternative point of view, our recent work sheds light on the role of an endocytosis-dependent calcium release from the endoplasmic reticulum in the control of nuclear cAMP levels. This is achieved through the activation of local soluble adenylyl cyclase, which in turn regulates the activation of local protein kinase A (PKA) and downstream transcriptional events. In this review, we explore the dynamic evolution of research on cyclic AMP signaling, including the findings that led us to formulate the novel three-wave hypothesis. We delve into how we abandoned the paradigm of cAMP generation limited to the plasma membrane and the changing perspectives on the rate-limiting step in nuclear PKA activation.

The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils.

Norbin (Neurochondrin, NCDN) is a GPCR adaptor protein known for its importance in neuronal function. Norbin works by binding to numerous GPCRs, controlling their steady state trafficking and sometimes their agonist-induced internalisation, as well as their signalling. We recently showed that Norbin is expressed in neutrophils, limits the surface levels of the GPCRs C5aR1 and CXCR4 in neutrophils, and suppresses neutrophil-mediated innate immunity. Here, we identify C5aR1 and CXCR4 as direct Norbin interactors and used mice with myeloid-Norbin deficiency to investigate the role of Norbin in the trafficking of endogenous C5aR1 and CXCR4 in primary neutrophils by flow cytometry and cell fractionation. We show that Norbin mediates the agonist-induced internalisation of C5aR1 through a ß-arrestin-dependent mechanism and limits the recycling of internalised C5aR1 and CXCR4 back to the cell surface. Norbin does not control the constitutive internalisation of C5aR1 and CXCR4, nor does it affect the agonist-induced internalisation of CXCR4. Norbin suppresses C5aR1 signalling in mouse neutrophils by limiting the C5a-stimulated membrane translocation of Tiam1, Vav, and PKCδ, and activation of Erk and p38 Mapk pathways, as well as Gαi-dependent ROS production. Our study demonstrates how Norbin suppresses C5aR1 and CXCR4 function in neutrophils and increases our understanding of the mechanisms through which Norbin regulates GPCR trafficking generally, by identifying its importance in ß-arrestin recruitment, ß-arrestin dependent agonist-induced receptor internalisation, and receptor recycling.

Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Structural analysis of the human C5a-C5aR1 complex using cryo-electron microscopy.

The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of ß-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.

Diversification processes of teleost intron-less opsin genes.

Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes that are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon, and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after the branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes in the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Hydrophobic interaction between the TM1 and H8 is essential for rhodopsin trafficking to vertebrate photoreceptor outer segments.

A major unsolved question in vertebrate photoreceptor biology is the mechanism of rhodopsin transport to the outer segment. In rhodopsin-like class A G protein-coupled receptors, hydrophobic interactions between C-terminal α-helix 8 (H8), and transmembrane α-helix-1 (TM1) have been shown to be important for transport to the plasma membrane, however whether this interaction is important for rhodopsin transport to ciliary rod outer segments is not known. We examined the crystal structures of vertebrate rhodopsins and class A G protein-coupled receptors and found a conserved network of predicted hydrophobic interactions. In Xenopus rhodopsin (xRho), this interaction corresponds to F313, L317, and L321 in H8 and M57, V61, and L68 in TM1. To evaluate the role of H8-TM1 hydrophobic interactions in rhodopsin transport, we expressed xRho-EGFP where hydrophobic residues were mutated in Xenopus rods and evaluated the efficiency of outer segment enrichment. We found that substituting L317 and M57 with hydrophilic residues had the strongest impact on xRho mislocalization. Substituting hydrophilic amino acids at positions L68, F313, and L321 also had a significant impact. Replacing L317 with M resulted in significant mislocalization, indicating that the hydrophobic interaction between residues 317 and 57 is exquisitely sensitive. The corresponding experiment in bovine rhodopsin expressed in HEK293 cells had a similar effect, showing that the H8-TM1 hydrophobic network is essential for rhodopsin transport in mammalian species. Thus, for the first time, we show that a hydrophobic interaction between H8 and TM1 is critical for efficient rhodopsin transport to the vertebrate photoreceptor ciliary outer segment.

The Wnt pathway protein Dvl1 targets somatostatin receptor 2 for lysosome-dependent degradation.

The Somatostatin receptor 2 (Sstr2) is a heterotrimeric G protein-coupled receptor that is highly expressed in neuroendocrine tumors and is a common pharmacological target for intervention. Unfortunately, not all neuroendocrine tumors express Sstr2, and Sstr2 expression can be downregulated with prolonged agonist use. Sstr2 is rapidly internalized following agonist stimulation and, in the short term, is quantitatively recycled back to the plasma membrane. However, mechanisms controlling steady state expression of Sstr2 in the absence of agonist are less well described. Here, we show that Sstr2 interacts with the Wnt pathway protein Dvl1 in a ligand-independent manner to target Sstr2 for lysosomal degradation. Interaction of Sstr2 with Dvl1 does not affect receptor internalization, recycling, or signaling to adenylyl cyclase but does suppress agonist-stimulated ERK1/2 activation. Importantly, Dvl1-dependent degradation of Sstr2 can be stimulated by overexpression of Wnts and treatment of cells with Wnt pathway inhibitors can boost Sstr2 expression in neuroendocrine tumor cells. Taken together, this study identifies for the first time a mechanism that targets Sstr2 for lysosomal degradation that is independent of Sstr2 agonist and can be potentiated by Wnt ligand. Intervention in this signaling mechanism has the potential to elevate Sstr2 expression in neuroendocrine tumors and enhance Sstr2-directed therapies.

Actin-binding protein filamin B regulates the cell-surface retention of endothelial sphingosine 1-phosphate receptor 1.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of ∼1 µM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed ß2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.