The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Proximity Interactome Analysis of Super Conserved Receptors Expressed in the Brain Identifies EPB41L2, SLC3A2, and LRBA as Main Partners.

The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the ß-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

Chemerin plasma levels are increased in COVID-19 patients and are an independent risk factor of mortality.

Background: Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value. Methods: Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels - Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC). Results: 21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS). Discussion: Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia.

The Atypical Chemerin Receptor GPR1 Displays Different Modes of Interaction with ß-Arrestins in Humans and Mice with Important Consequences on Subcellular Localization and Trafficking.

Atypical chemokine receptors (ACKRs) have emerged as a subfamily of chemokine receptors regulating the local bioavailability of their ligands through scavenging, concentration, or transport. The biological roles of ACKRs in human physiology and diseases are often studied by using transgenic mouse models. However, it is unknown whether mouse and human ACKRs share the same properties. In this study, we compared the properties of the human and mouse atypical chemerin receptor GPR1 and showed that they behave differently regarding their interaction with ß-arrestins. Human hGPR1 interacts with ß-arrestins as a result of chemerin stimulation, whereas its mouse orthologue mGPR1 displays a strong constitutive interaction with ß-arrestins in basal conditions. The constitutive interaction of mGPR1 with ß-arrestins is accompanied by a redistribution of the receptor from the plasma membrane to early and recycling endosomes. In addition, ß-arrestins appear mandatory for the chemerin-induced internalization of mGPR1, whereas they are dispensable for the trafficking of hGPR1. However, mGPR1 scavenges chemerin and activates MAP kinases ERK1/2 similarly to hGPR1. Finally, we showed that the constitutive interaction of mGPR1 with ß-arrestins required different structural constituents, including the receptor C-terminus and arginine 3.50 in the second intracellular loop. Altogether, our results show that sequence variations within cytosolic regions of GPR1 orthologues influence their ability to interact with ß-arrestins, with important consequences on GPR1 subcellular distribution and trafficking.

Chemerin regulates normal angiogenesis and hypoxia-driven neovascularization.

Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral factor inhibiting the vascularization of tumor grafts. We show here that overexpression of bioactive chemerin in mice results in a reduction of the density of the retinal vascular network during its development and in adults. Chemerin did not affect vascular sprouting during the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating the role of this receptor. Chemerin inhibited also neoangiogenesis in a model of pathological proliferative retinopathy, and in response to hind-limb ischemia. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the density of the retinal vasculature, suggesting the involvement of the PI3-kinase/AKT pathway in the chemerin-induced vessel regression process.

Expression of CCRL2 Inhibits Tumor Growth by Concentrating Chemerin and Inhibiting Neoangiogenesis.

CCRL2 belongs to the G protein-coupled receptor family and is one of the three chemerin receptors. It is considered as a non-signaling receptor, presenting chemerin to cells expressing the functional chemerin receptor ChemR23/CMKLR1 and possibly GPR1. In the present work, we investigate the role played by CCRL2 in mouse cancer models. Loss of function of Ccrl2 accelerated the development of papillomas in a chemical model of skin carcinogenesis (DMBA/TPA), whereas the growth of B16 and LLC tumor cell grafts was delayed. Delayed tumor growth was also observed when B16 and LLC cells overexpress CCRL2, while knockout of Ccrl2 in tumor cells reversed the consequences of Ccrl2 knockout in the host. The phenotypes associated with CCRL2 gain or loss of function were largely abrogated by knocking out the chemerin or Cmklr1 genes. Cells harboring CCRL2 could concentrate bioactive chemerin and promote the activation of CMKLR1-expressing cells. A reduction of neoangiogenesis was observed in tumor grafts expressing CCRL2, mimicking the phenotype of chemerin-expressing tumors. This study demonstrates that CCRL2 shares functional similarities with the family of atypical chemokine receptors (ACKRs). Its expression by tumor cells can significantly tune the effects of the chemerin/CMKLR1 system and act as a negative regulator of tumorigenesis.

TAFA4 relieves injury-induced mechanical hypersensitivity through LDL receptors and modulation of spinal A-type K+ current.

Pain, whether acute or persistent, is a serious medical problem worldwide. However, its management remains unsatisfactory, and new analgesic molecules are required. We show here that TAFA4 reverses inflammatory, postoperative, and spared nerve injury (SNI)-induced mechanical hypersensitivity in male and female mice. TAFA4 requires functional low-density lipoprotein receptor-related proteins (LRPs) because their inhibition by RAP (receptor-associated protein) dose-dependently abolishes its antihypersensitive actions. SNI selectively decreases A-type K+ current (IA) in spinal lamina II outer excitatory interneurons (L-IIo ExINs) and induces a concomitant increase in IA and decrease in hyperpolarization-activated current (Ih) in lamina II inner inhibitory interneurons (L-IIi InhINs). Remarkably, SNI-induced ion current alterations in both IN subtypes were rescued by TAFA4 in an LRP-dependent manner. We provide insights into the mechanism by which TAFA4 reverses injury-induced mechanical hypersensitivity by restoring normal spinal neuron activity and highlight the considerable potential of TAFA4 as a treatment for injury-induced mechanical pain.

The antitumoral effects of chemerin are independent from leukocyte recruitment and mediated by inhibition of neoangiogenesis.

Chemerin, a multifunctional protein acting through the receptor ChemR23/CMKLR1, is downregulated in various human tumors and was shown to display antitumoral properties in mouse models of cancer. In the present study, we report that bioactive chemerin expression by tumor cells delays the growth of B16 melanoma and Lewis lung carcinoma in vivo. A similar delay is observed when chemerin is not expressed by tumor cells but by keratinocytes of the host mice. The protective effect of chemerin is mediated by CMKLR1 and appears unrelated to the recruitment of leukocyte populations. Rather, tumors grown in the presence of chemerin display a much smaller number of blood vessels, hypoxic regions early in their development, and larger necrotic areas. These observations likely explain the slower growth of the tumors. The anti-angiogenic effects of chemerin were confirmed in a bead sprouting assay using human umbilical vein endothelial cells. These results suggest that CMKLR1 agonists might constitute therapeutic molecules inhibiting the neoangiogenesis process in solid tumors.

CCRL2 Modulates Physiological and Pathological Angiogenesis During Retinal Development.

Chemerin is a multifunctional protein involved in the regulation of inflammation, metabolism, and tumorigenesis. It binds to three receptors, CMKLR1, GPR1 and CCRL2. CMKLR1 is a fully functional receptor mediating most of the known activities of chemerin. CCRL2 does not seem to couple to any intracellular signaling pathway and is presently considered as an atypical receptor able to present the protein to cells expressing CMKLR1. CCRL2 is expressed by many cell types including leukocyte subsets and endothelial cells, and its expression is strongly upregulated by inflammatory stimuli. We recently reported that chemerin can negatively regulate the angiogenesis process, including during the development of the vascular network in mouse retina. The role of CCRL2 in angiogenesis was unexplored so far. In the present work, we demonstrate that mice lacking CCRL2 exhibit a lower density of vessels in the developing retina and this phenotype persists in adulthood, in a CMKLR1-dependent manner. Vascular sprouting was not affected, while vessel pruning, and endothelial cell apoptosis were increased. Pathological angiogenesis was also reduced in CCRL2-/- mice in a model of oxygen-induced retinopathy. The phenotype closely mimics that of mice overexpressing chemerin, and the concentration of chemerin was found elevated in the blood of newborn mice, when the retinal vasculature develops. CCRL2 appears therefore to regulate the distribution and concentration of chemerin in organs, regulating thereby its bioactivity.

Corrigendum: Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

[This corrects the article DOI: 10.3389/fonc.2019.01253.].

Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

Chemerin is a multifunctional protein acting mainly through the G protein-coupled receptor ChemR23/CMKLR1/Chemerin1. Its expression is frequently downregulated in human tumors, including in melanoma and squamous cell carcinoma of the skin and anti-tumoral properties of chemerin were reported in mouse tumor graft models. In the present study, we report the development of spontaneous skin tumors in aged ChemR23-deficient mice. In order to test the potential therapeutic benefit of chemerin analogs, a transgenic model in which bioactive chemerin is over-expressed by basal keratinocytes was generated. These animals are characterized by increased levels of chemerin immunoreactivity and bioactivity in the skin and the circulation. In a chemical carcinogenesis model, papillomas developed later, were less numerous, and their progression to carcinomas was delayed. Temporal control of chemerin expression by doxycycline allowed to attribute its effects to late stages of carcinogenesis. The protective effects of chemerin were partly abrogated by ChemR23 invalidation. These results demonstrate that chemerin is able to delay very significantly tumor progression in a model that recapitulates closely the evolution of solid cancer types in human and suggest that the chemerin-ChemR23 system might constitute an interesting target for therapeutic intervention in the cancer field.

Coexpression of CCR7 and CXCR4 During B Cell Development Controls CXCR4 Responsiveness and Bone Marrow Homing.

The CXCL12-CXCR4 axis plays a key role in the retention of stem cells and progenitors in dedicated bone marrow niches. It is well-known that CXCR4 responsiveness in B lymphocytes decreases dramatically during the final stages of their development in the bone marrow. However, the molecular mechanism underlying this regulation and whether it plays a role in B-cell homeostasis remain unknown. In the present study, we show that the differentiation of pre-B cells into immature and mature B cells is accompanied by modifications to the relative expression of chemokine receptors, with a two-fold downregulation of CXCR4 and upregulation of CCR7. We demonstrate that expression of CCR7 in B cells is involved in the selective inactivation of CXCR4, and that mature B cells from CCR7-/- mice display higher responsiveness to CXCL12 and improved retention in the bone marrow. We also provide molecular evidence supporting a model in which upregulation of CCR7 favors the formation of CXCR4-CCR7 heteromers, wherein CXCR4 is selectively impaired in its ability to activate certain G-protein complexes. Collectively, our results demonstrate that CCR7 behaves as a novel selective endogenous allosteric modulator of CXCR4.

Development by Genetic Immunization of Monovalent Antibodies Against Human Vasoactive Intestinal Peptide Receptor 1 (VPAC1), New Innovative, and Versatile Tools to Study VPAC1 Receptor Function.

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions.

Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges.

Le STUDIUM conference was held November 24-25, 2016 in Tours, France as a satellite workshop of the 5th meeting of the French GDR 3545 on "G Protein-Coupled Receptors (GPCRs) -From Physiology to Drugs," which was held in Tours during November 22-24, 2016. The conference gathered speakers from academia and industry considered to be world leaders in the molecular pharmacology and signaling of GPCRs, with a particular interest in the development of therapeutic GPCR antibodies (Abs). The main topics were new advances and challenges in the development of antibodies targeting GPCRs and their potential applications to the study of the structure and function of GPCRs, as well as their implication in physiology and pathophysiology. The conference included 2 sessions, with the first dedicated to the recent advances in methodological strategies used for GPCR immunization using thermo-stabilized and purified GPCRs, and the development of various formats of Abs such as monoclonal IgG, single-chain variable fragments and nanobodies (Nbs) by in vitro and in silico approaches. The second session focused on GPCR Nbs as a "hot" field of research on GPCRs. This session started with discussion of the pioneering Nbs developed against GPCRs and their application to structural studies, then transitioned to talks on original ex vivo and in vivo studies on GPCR-selective Nbs showing promising therapeutic applications of Nbs in important physiologic systems, such as the central nervous and the immune systems, as well as in cancer. The conference ended with the consensus that Abs and especially Nbs are opening a new era of research on GPCR structure, pharmacology and pathophysiology.

Truncation of CXCL12 by CD26 reduces its CXC chemokine receptor 4- and atypical chemokine receptor 3-dependent activity on endothelial cells and lymphocytes.

The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH2-terminal amino acids. CD26-truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3-68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3-68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP3), Akt or extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of ß-arrestin 2 to the cell membrane via CXCR4 by CXCL12(3-68) was abolished, whereas a weakened but significant ß-arrestin recruitment remained via ACKR3. CXCL12-induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra-articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra-articular lymphocyte infiltration was significantly increased in sitagliptin-treated mice and CXCL12(3-68) failed to induce migration under both CD26-inhibiting and non-inhibiting conditions. In conclusion, CD26-cleavage skews CXCL12 towards ß-arrestin dependent recruitment through ACKR3 and destroys the CXCR4-mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26-blockade in tissues may enhance CXCL12-induced inflammation.

Complete removal of arsenic and zinc from a heavily contaminated acid mine drainage via an indigenous SRB consortium.

Acid mine drainages (AMD) are major sources of pollution to the environment. Passive bio-remediation technologies involving sulfate-reducing bacteria (SRB) are promising for treating arsenic contaminated waters. However, mechanisms of biogenic As-sulfide formation need to be better understood to decontaminate AMDs in acidic conditions. Here, we show that a high-As AMD effluent can be decontaminated by an indigenous SRB consortium. AMD water from the Carnoulès mine (Gard, France) was incubated with the consortium under anoxic conditions and As, Zn and Fe concentrations, pH and microbial activity were monitored during 94days. Precipitated solids were analyzed using electron microscopy (SEM/TEM-EDXS), and Extended X-Ray Absorption Fine Structure (EXAFS) spectroscopy at the As K-edge. Total removal of arsenic and zinc from solution (1.06 and 0.23mmol/L, respectively) was observed in two of the triplicates. While Zn precipitated as ZnS nanoparticles, As precipitated as amorphous orpiment (am-AsIII2S3) (33-73%), and realgar (AsIIS) (0-34%), the latter phase exhibiting a particular nanowire morphology. A minor fraction of As is also found as thiol-bound AsIII (14-23%). We propose that the formation of the AsIIS nanowires results from AsIII2S3 reduction by biogenic H2S, enhancing the efficiency of As removal. The present description of As immobilization may help to set the basis for bioremediation strategies using SRB.

Partial Agonist and Biased Signaling Properties of the Synthetic Enantiomers J113863/UCB35625 at Chemokine Receptors CCR2 and CCR5.

Biased agonism at G protein-coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12 Binding of J113863 to CCR2 or CCR5 also induced the recruitment of ß-arrestin 2, whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and ß-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist, or full agonist according to the receptor, the enantiomer, and the signaling pathway investigated.

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of ß-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate ß-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and ß-arrestin2 activation but not ß-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.