Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.

The sensory neuron-specific G protein coupled receptors (SNSRs) have been described as a family of receptors whose expression in small diameter sensory neurons in the trigeminal and dorsal root ganglia suggests an implication in nociception. To date, the physiological function(s) of SNSRs remain unknown. Hence, the aim of the present study was to determine the effects of rat SNSR1 activation on nociception in rats. The pharmacological characterization of rat SNSR1 was initially performed in vitro to identify a specific ligand, which could be used subsequently in the rat for physiological testing. Among all ligands tested, ␥2-MSH was the most potent at activating rat SNSR1. Structure-activity relationship studies revealed that the active moiety recognized by rat SNSR1 was the C-terminal part of ␥2-MSH. The radiolabeled C-terminal part of ␥2-MSH, ␥2-MSH-6 -12, bound with high affinity to membranes derived from rat skin and spinal cord, demonstrating the presence of receptor protein at both the proximal and distal terminals of dorsal root ganglia. To investigate the physiological role of SNSR, specific ligands to rat SNSR1 were tested in behavioral assays of pain sensitivity in rats. Selective rat SNSR1 agonists produced spontaneous pain behavior, enhanced heat and mechanical sensitivity when injected intradermally, and heat hypersensitivity when injected centrally, consistent with the localization of rat SNSR1 protein at central and peripheral sites. Together, these results clearly indicate that the SNSR1 plays a role in nociception and may provide novel therapeutic opportunities for analgesia. Gprotein-coupled receptors constitute one of the largest gene family of proteins that have been exploited successfully as drug targets (1). With the near completion of the human genome project, bioinformatic analyses have revealed the existence of Ϸ145 orphan receptors (1, 2). In recent years, the ''reverse pharmacology approach'' has generated Ͼ40 ligand-receptor pairings (1, 3), and the subsequent investigation of these newly discovered ligand-receptor pairings should help in understanding and elucidating their potential physiological and pathophysiological roles.We cloned a previously undescribed family of G protein-coupled receptors that we named the sensory neuron-specific receptors (SNSRs) because of their unique mRNA distribution in small nociceptive sensory neurons in dorsal root (DRG) and trigeminal ganglia (4). The SNSRs are phylogenetically related to the Mas oncogene receptor and belong to the Mas-related genes or Mrg family described for mouse and human by Dong et al. (5). Based on several analyses, this subfamily of receptors is comprised of four to six members in human (MrgX1-4 or SNSR1-6) and 32 receptors in mouse classified into three major subfamilies Mrg A, B, and C (4-7). Initially, only one SNSR gene was identified in rat (4); recently, Zylka et al. (8) have demonstrated that more than one rat SNSR͞ Mrg subtype exists. These receptors have been subclassified in a similar scheme as described for human a...

Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11bhiCD103− and CD11bloCD103+ dendritic cells (DCs), and CD4+ and CD8+ T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11bhiCD103− DCs, and activated CD4+ T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8+ T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.