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.

Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor?

G protein-coupled receptors (GPCRs) are membrane-bound receptors that are considered prime candidates for the development of novel insect pest management strategies. However, the molecular signaling properties of insect GPCRs remain poorly understood. In fact, most studies on insect GPCR signaling are limited to analysis of fluctuations in the secondary messenger molecules calcium (Ca2+) and/or cyclic adenosine monophosphate (cAMP). In the current study, we characterized a corticotropin-releasing factor-related diuretic hormone (CRF-DH) receptor of the desert locust, Schistocerca gregaria. This Schgr-CRF-DHR is mainly expressed in the nervous system and in brain-associated endocrine organs. The neuropeptide Schgr-CRF-DH induced Ca2+-dependent aequorin-based bioluminescent responses in CHO cells co-expressing this receptor with the promiscuous Gα16 protein. Furthermore, when co-expressed with the cAMP-dependent bioluminescence resonance energy transfer (BRET)-based CAMYEL biosensor in HEK293T cells, this receptor elicited dose-dependent agonist-induced responses with an EC50 in the nanomolar range (4.02 nM). In addition, we tested if vertebrate BRET-based G protein biosensors, can also be used to detect direct Gα protein subunit activation by an insect GPCR. Therefore, we analyzed ten different human BRET-based G protein biosensors, representing members of all four Gα protein subfamilies; Gαs, Gαi/o, Gαq/11 and Gα12/13. Our data demonstrate that stimulation of Schgr-CRF-DHR by Schgr-CRF-DH can dose-dependently activate Gαi/o and Gαs biosensors, while no significant effects were observed with the Gαq/11 and Gα12/13 biosensors. Our study paves the way for future biosensor-based studies to analyze the signaling properties of insect GPCRs in both fundamental science and applied research contexts.

Chemerin influences endothelin- and serotonin-induced pulmonary artery vasoconstriction in rats.

AIMS: Chemerin has been recently identified as a vasoactive adipokine implicated in blood pressure regulation. In this context, we evaluated whether chemerin could influence pulmonary vasoreactive response. MATERIALS AND METHODS: Vascular reactivity to chemerin and to phenylephrine, serotonin and endothelin-1 after chemerin pretreatment was evaluated in rat isolated pulmonary artery versus thoracic aorta with and without endothelium. Vasoreactivity to acetylcholine in presence of nitric oxide (NO)-synthase inhibitor (L-NAME) and to NO donor sodium nitroprusside (SNP) was evaluated in chemerin-pretreated pulmonary artery versus thoracic aorta with endothelium. Pretreatment with ODQ, a soluble guanylate cyclase inhibitor and apocynin, a ROS production inhibitor, were also tested. Arteries and lung tissue were harvested for pathobiological evaluation. KEY FINDINGS: Chemerin contracted endothelium-denuded pulmonary artery, while no response was observed in arteries with endothelium. Chemerin potentiated phenylephrine-, endothelin-1- and serotonin-induced vasoconstriction, which was further enhanced by endothelium removal. Chemerin decreased acetylcholine-induced vasorelaxation in arteries with endothelium, while it did not affect SNP-induced relaxation. In presence of L-NAME, there remained a vasorelaxation in chemerin-pretreated arteries. Chemerin or ODQ alone partly decreased acetylcholine-induced vasorelaxation in pulmonary artery and thoracic aorta, while combined chemerin and ODQ incubation abolished it. Treatment with apocynin partly or totally reversed chemerin effects. In both types of arteries, chemerin reduced acetylcholine-induced NO production, as well as endothelial and inducible NO-synthase expression. SIGNIFICANCE: Chemerin potentiates vascular responses to vasoconstrictors in pulmonary artery and thoracic aorta and, impairs acetylcholine-induced pulmonary artery vasodilatation, by mechanisms involving at least partly NO signaling and oxidative stress.

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.