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GPRC6A (GPCR, class C, group 6, subtype A) is a class C GPCR that has been cloned from human, mouse and rat. Several groups have shown that the receptor is activated by a range of basic and small aliphatic L-α-amino acids of which L-arginine, L-lysine and L-ornithine are the most potent compounds with EC50 values in the mid-micromolar range. In addition, several groups have shown that the receptor is either directly activated or positively modulated by divalent cations such as Ca2+ albeit in concentrations above 5 mM, which is above the physiological concentration in most tissues. More recently, the peptide osteocalcin and the steroid testosterone have also been suggested to be endogenous GPRC6A agonists. The receptor is widely expressed in all three species which, along with the omnipresence of the amino acids and divalent cation ligands, suggest that the receptor could be involved in a broad range of physiological functions. So far, this has mainly been addressed by analyses of genetically modified mice where the GPRC6A receptor has been ablated. Although there has been some discrepancies among results reported from different groups, there is increasing evidence that the receptor is involved in regulation of inflammation, metabolism and endocrine functions. GPRC6A could thus be an interesting target for new drugs in these therapeutic areas. Linked Articles This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [(3)H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Purinergic receptors, also known as purinoceptors, are ligand gated membrane ion channels involved in many cellular functions. Among all identified purinergic receptors, P2X₇ subform is unique since it induces the caspase activity, cytokine secretion, and apoptosis. The distribution of P2X₇ receptors, and the need of high concentration of ATP required to activate this receptor exhibited its ability to function as 'danger' sensor associated with tissue inflammation and damage. Further, the modulation of other signalling pathways associated with P2X₇ has also been proposed to play an important role in the control of macrophage functions and inflammatory responses, especially towards lipopolysaccharides. Experimentally, researchers have also observed the decreased severity of inflammatory responses in P2X₇ receptor expressing gene (P2RX₇) knockout (KO) phenotypes. Therefore, newly developed potent antagonists of P2X₇ receptor would serve as novel therapeutic agents to combat various inflammatory conditions. In this review article, we tried to explore various aspects of P2X₇ receptors including therapeutic potential, and recent discoveries and developments of P2X₇ receptor antagonists.

International audience; Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn 2+ , Co 2+ or Cu 2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co 2+ , Cd 2+ or Cu 2+ . Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level.

A class of α-methyltryptamine sulfonamide glucocorticoid receptor (GR) modulators was optimized for agonist activity. The design of ligands was aided by molecular modeling, and key function-regulating pharmacophoric points were identified that are critical in achieving the desired agonist effect in cell based assays. Compound 27 was profiled in vitro and in vivo in models of inflammation. Analogs could be rapidly prepared in a parallel approach from aziridine building blocks.

The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49(1.30) to M76(1.57)) and TM2 (V88(2.41) to H117(2.70)) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of 125I-UII to mutant I54C(1.35) in TM1 and mutants Y100C(2.53), S103C(2.56), F106C(2.59), I107C(2.60), T110C(2.63) and Y111C(2.64) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of β2-adrenergic receptor (β2AR) in complex with Gs (β2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-2183.54, Tyr-224IC2, Asp-3386.30, Arg-3406.32, Leu-3416.33, and Thr-3446.36, as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation. Background: The molecular basis of CB1 coupling to its cognate G protein is unknown. Results: Using an approach combining mutagenesis and molecular dynamics simulations, we identified CB1 residues critical for G protein signaling. Conclusion: Tight interactions between CB1 and the C-terminal helix α5 of Gαi are crucial for G protein signaling. Significance: This is the first reported molecular description of CB1 receptor coupling at the receptor-Gi interface.

Background and Purpose In addition to typical GPCR kinase (GRK)-/β-arrestin-dependent internalization, dopamine D3 receptor employed an additional GRK-independent sequestration pathway. In this study, we investigated the molecular mechanism of this novel sequestration pathway. Experimental Approach Radioligand binding, flow cytometry and cell surface biotinylation assay were used to characterize trafficking properties of D2 and D3 receptors. Serine/threonine and N-linked glycosylation mutants of the D3 receptor were utilized to locate receptor regions involved in pharmacological sequestration and desensitization. Various point mutants of the D2 and D3 receptors, whose sequestration and desensitization properties were altered, were combined with knockdown cells of GRKs or β-arrestins to functionally correlate pharmacological sequestration and desensitization. Key Results The D3 receptor, but not the D2 receptor, showed characteristic trafficking behaviour in which receptors were shifted towards the more hydrophobic domains within the plasma membrane without translocation into other intracellular compartments. Among various amino acid residues tested, S145/S146, C147 and N12/19 were involved in pharmacological sequestration and receptor desensitization. Both pharmacological sequestration and desensitization of D3 receptor required β-arrestins, and functional relationship was observed between two processes when it was tested for D3 receptor variants and agonists. Conclusions and Implications Pharmacological sequestration of D3 receptor accompanies movement of cell surface receptors into a more hydrophobic fraction within the plasma membrane and renders D3 receptor inaccessible to hydrophilic ligands. Pharmacological sequestration is correlated with desensitization of the D3 receptor in a Gβγ- and β-arrestin-dependent manner. This study provides new insights into molecular mechanism governing GPCR trafficking and desensitization.

A series of heterocyclic glucocorticoid receptor (GR) modulators with 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl)propanamide core are described. Structure-activity relationships suggest a combination of H-bond acceptor and a 4-fluorophenyl moiety as being important structural components contributing to the glucocorticoid receptor binding and functional activity for this series of GR modulators.

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine-piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

Background and Purpose Since the identification of the histamine H4 receptor, several ligands activating this receptor have been described and more compounds are in development. These ligands are well characterized in pharmacological assays, including radioligand competition binding studies, GTPγS and GTPase assays. In most cases, these experiments are performed in transfected cell lines, expressing unnaturally high levels of target receptors and G-protein signalling components. In this study we investigated the specific properties of H4 receptor ligands in native cells. Experimental Approach Histamine and five different H4 receptor agonists – 4-methylhistamine, UR-PI376, clobenpropit, VUF8430 and ST-1006 – were characterized in freshly isolated human monocytes. The ligands (10 nM–10 μM) were tested as inhibitors of IL-12p70 secretion from human monocytes and the effects of the H2 receptor antagonist ranitidine and the H4 receptor antagonist JNJ7777120 on their action was investigated. Key Results Histamine and all the tested agonists reduced IL-12p70 secretion into monocyte supernatants by 40–70%. The potencies varied with pEC50 values ranging from 5.7 to 6.9, depending on the agonist used. All potencies were lower than those determined in the original investigations of the compounds. Pretreatment of monocytes with H2 or H4 receptor antagonists showed that some H4 receptor ligands also had low activity at the H2 receptor. Conclusions and Implications Our study demonstrates discrepancies between the potencies obtained from assays in transfected cell lines and assays in native human cells, indicating the importance of evaluating H4 receptor ligands in native cells. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.

The cytokine IL-1 is critical to the pathogenesis of a variety of human conditions and diseases. Unlike most other cytokines, IL-1 is counterbalanced by two endogenous inhibitors. The functional significance of IL-1 receptor antagonist (IL-1RA) is well documented due to the clinical utilization of the recombinant human IL-1RA analog, anakinra. In contrast, much less is known about the type 2 IL-1 receptor (IL-1R2), which acts as a decoy receptor for IL-1. While IL-1R2 is structurally similar to the type 1 IL-1 receptor (IL-1R1) responsible for IL-1 signal transduction, its truncated cytoplasmic domain and lack of Toll-IL-1 receptor (TIR) region renders IL-1R2 incapable of transmembrane signaling. IL-1R2 competes with IL-1R1 for ligands and for the IL-1R1 co-receptor, IL-1 receptor accessory protein (IL-1RAP). Additionally, IL-1R2 exists in both a membrane bound and soluble form (sIL-1R2) that has biological properties similar to both a decoy receptor and a binding protein. Thus far, IL-1R2 has been implicated in arthritis, endometriosis, organ transplantation, sepsis/sickness behavior, diabetes, atherosclerosis, autoimmune inner ear disease (AIED), Alzheimer’s disease and ulcerative colitis. In this review, we will detail the functional properties of IL-1R2 and examine its role in human disease.

The pleiotropic pro-inflammatory cytokine interleukin (IL)-1β has co-evolved with a competitive inhibitor, IL-1 receptor antagonist (IL-1Ra). IL-1β initiates cell signaling by binding the IL-1 receptor (IL-1R) whereas IL-1Ra acts as an antagonist, blocking receptor signaling. The current paradigm for agonist/antagonist functions for these two proteins is based on the receptor–ligand interaction observed in the crystal structures of the receptor–ligand complexes. While IL-1Ra and IL-1β are structurally homologous, IL-1Ra engages only two of the three extracellular domains of the receptor, whereas IL-1β engages all three. We find that an allosteric functional switch exists within a highly conserved pocket of residues, residues 111–120. This region is maintained across all IL-1 family members and serves as a hydrophobic mini-core for IL-1β folding. A key difference across species is a conserved aromatic residue at position 117 in IL-1β, versus a conserved cysteine in IL-1Ra at the analogous position, 116. We find that the replacement of C116 with a phenylalanine switches the protein from an antagonist to an agonist despite the distant location of C116 relative to receptor interaction sites. These results suggest new ways to develop designer cytokine activity into the β-trefoil fold and may be of general use in regulation of this large family of signaling proteins.

The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1-MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.

The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists.

Background and Purpose The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. Experimental Approach 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor. Linked Articles Recent reviews on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476-5381.2012.02223.x Roche D, Gil D and Giraldo J (2013). Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two-state and operational models. British Journal of Pharmacology 169: 1189–1202. doi: 10.1111/bph.12231

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 μM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

The D3 dopamine receptor is a therapeutic target for treating various nervous system disorders such as schizophrenia, Parkinson's disease, depression, and addictive behaviors. The crystal structure of the D3 receptor bound to an antagonist was recently described; however, the structural features that contribute to agonist-induced conformational changes and signaling properties are not well understood. We have previously described the conformation-dependent tolerance and slow response termination (SRT) signaling properties of the D3 receptor and identified the C147 residue in the second intracellular loop (IL2) of the D3 receptor as important for the tolerance property. Interestingly, while IL2 and the C147 residue, in particular, were important for dopamine- and quinpirole-induced tolerance, this residue did not affect the severe tolerance induced by the high affinity, D3 receptor-selective agonist, PD128907. Here, we used D2/D3 receptor chimeras and site-specific D3 receptor mutants to identify another residue, D187, in the second extracellular loop (EC2) of the human D3 receptor that mediates the tolerance property induced by PD128907, quinpirole, pramipexole, and dopamine. Molecular dynamics simulations confirmed the distinct conformation adopted by D3 receptor during tolerance and suggested that in the tolerant D3 receptor the D187 residue in EC2 forms a salt bridge with the H354 residue in EC3. Indeed, site-directed mutation of the H354 residue resulted in loss of PD1287907-induced tolerance. The mapping of specific amino acid residues that contribute to agonist-dependent conformation changes and D3 receptor signaling properties refines the agonist-bound D3 receptor pharmacophore model which will help develop novel D3 receptor agonists.

IL-33 (IL-1F11) is a member of IL-1 family ligand, which stimulates the production of inflammatory cytokines. IL-33 receptor complex is comprised of IL-1 receptor accessory protein (IL-1RAcP) and ST2 that are activated by IL-33 ligand binding. ST2 is a ligand-binding chain of the IL-33 receptor component, and the soluble ST2 form possesses antagonistic activity. Here, we expressed the extracellular domain of ST2-fused to the immunoglobulin of IgG1 constant region in order to generate a soluble recombinant Fc-ST2. Human and mouse recombinant Fc-ST2 protein were expressed in Chinese hamster ovary cells and purified using a mini-protein A affinity chromatography. The recombinant Fc-ST2 protein was used to examine inhibitory function in IL-33-induced cytokine production in different cell types. The human Fc-ST2 abolished IL-33-induced IL-8 production in human mast cells, but mouse Fc-ST2 failed to inhibit IL-33-induced TNFα production in mouse Raw 264.7 macrophage cells. We further investigated the expression of IL-33 receptor component with various cell lines. IL-33 receptors expression pattern and Fc-ST2 inhibitory activity in different cell types suggest that IL-1RAcP and ST2 are necessary but insufficient for IL-33 activity. Our results suggest that an additional receptor component may participate in the biological activity of IL-33.

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is the fourth and most recently discovered member of the opioid receptor superfamily that also includes μ, δ, and κ opioid receptor subtypes (MOR, DOR, and KOR, respectively). The widespread anatomic distribution of the NOP receptor enables the modulation of several physiologic processes by its endogenous agonist, N/OFQ. Accordingly, the NOP receptor has gained a lot of attention as a potential target for the development of ligands with therapeutic use in several pathophysiological states. NOP receptor activation frequently results in effects opposing classic opioid receptor action; therefore, regulation of the NOP receptor and conditions affecting its modulatory tone are important to understand. Mounting evidence reveals a heterologous interaction of the NOP receptor with other G protein-coupled receptors, including MOR, DOR, and KOR, which may subsequently influence their function. Our focus in this review is to summarize and discuss the findings that delineate the cellular mechanisms of NOP receptor signaling and regulation and the regulation of other receptors by N/OFQ and the NOP receptor.

The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment.

SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 (D(1) receptor) agonist, has shown many D(1) receptor-independent effects, such as neuroprotection, blockade of Na(+) channel, and promotion of spontaneous glutamate release, which resemble the effects of the sigma-1 receptor activation. In the present work, we explored the potential modulation of SKF83959 on the sigma-1 receptor. The results indicated that SKF83959 dramatically promoted the binding of (3)H(+)-pentazocine (a selective sigma-1 receptor agonist) to the sigma-1 receptor in brain and liver tissues but produced no effect on (3)H-progesterone binding (a sigma-1 receptor antagonist). The saturation assay and the dissociation kinetics assay confirmed the allosteric effect. We further demonstrated that the SKF83959 analogs, such as SCH22390 [(R)-(1)-7-chloro-8- hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] and SKF38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], also showed the similar allosteric effect on the sigma-1 receptor in the liver tissue but not in the brain tissue. Moreover, all three tested chemicals elicited no significant effect on (3)H-1,3-di(2-tolyl)-guanidine ((3)H-DTG) binding to the sigma-2 receptor. The present data uncovered a new role of SKF83959 and its analogs on the sigma-1 receptor, which, in turn, may reveal the underlying mechanism for the D(1) receptor-independent effect of the drug.

The endothelin (ET) system consists of two G protein coupled–receptors (GPCRs), ET type A receptor (ETAR) and ET type B receptor (ETBR), and three endogenous ligands, ET-1, ET-2, and ET-3. Stimulation of ETRs with ET-1 induces an increase in intracellular Ca2+ concentration that is involved in a diverse array of physiological and pathophysiological processes, including vasoconstriction, and cell proliferation. Store-operated Ca2+ entry and receptor-operated Ca2+ entry triggered by activation of ETRs are regulated or modulated by endoplasmic reticulum Ca2+ sensor (stromal interaction molecule 1) and voltage-independent cation channels (transient receptor potential canonical channels and Orai1). The ET-1–induced Ca2+ mobilization results from activation of heterotrimeric G proteins by ETRs. In contrast, GPCR biology including modulation of receptor function and trafficking is regulated by a variety of GPCR interacting proteins (GIPs) that generally interact with the C-terminal domain of GPCRs. The ETR signaling is also regulated by GIPs such as Jun activation domain-binding protein 1. This review focuses on the regulatory mechanisms of the ETR signaling with special attention to the components involved in Ca2+ signaling and to GIPs in the signal transduction, modification, and degradation of ETRs. Keywords:: endothelin receptor, G protein–coupled receptor, G protein–coupled receptor interacting protein, Jun activation domain–binding protein 1, stromal interaction molecule

Histamine is a biogenic amine that exerts its biological effects as a neurotransmitter and local mediator via four histamine receptor (HR) subtypes (H(x)Rs) - H(1)R, H(2)R, H(3)R, and H(4)R - belonging to the superfamily of G-protein-coupled receptors (GPCRs). All four H(x)Rs exhibit pronounced differences in agonist and/or antagonist pharmacology among various species orthologs. The species differences constitute a problem for animal experiments and drug development. This problem applies to GPCRs with diverse ligands. Here, we summarize our current knowledge on H(x)R orthologs as a case study for species-dependent activity of GPCR ligands. We show that species-specific pharmacology also provides unique opportunities to study important aspects of GPCR pharmacology in general, including ligand-binding sites, the roles of extracellular domains in ligand binding and receptor activation, agonist-independent (constitutive) receptor activity, thermodynamics of ligand/receptor interaction, receptor-activation mechanisms, and ligand-specific receptor conformations.

When considering the interactions between the melanocortin peptides (i.e., ACTH, α-MSH, β-MSH, γ-MSH) and the melanocortin receptors (i.e., MC1R, MC2R, MC3R, MC4R, MC5R), it appears that the structure/function relationship between ACTH and MC2R is the most complicated. Human ACTH(1-24) and the human melanocortin-2 receptor provide a useful model system for understanding how ACTH emerged as the sole ligand for the melanocortin-2 receptor of bony vertebrates. This review will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for activation of the melanocortin-2 receptor. In addition, observations on the unique activation features of the melanocortin-2 receptor, as revealed from studies on Familial Glucocorticoid Deficiency, will be considered. Finally, the evolutionary implications of the relationship between MC2R and MRAP1 will be discussed.

El pdf del artículo es la versión post-print.-- et al., Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-6774.40, Ala-6814.44, and Ala-6854.48) leads to absence of 5-HT2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer., This work was supported, in whole or in part, by National Institutes of Health Grants R01 MH084894 (to J.G.M.), R01 NS37731 (to D.L.B.), and P01 DA12923 (to S.C.S.). This work was also supported by Dainippon Sumi-tomo Pharma (to J.G.M.), National Alliance for Research on Schizophrenia and Depression (to J.G.M.), The Mortimer D. Sackler Foundation (to J.G.M.), Ministerio de Ciencia e Innovación Grant SAF2009-084609 (to J.J.M.), Basque Government Grant IT-199-07 (to J.J. M.), Consejo Superior de Investigaciones Científicas Grants PA1003176 and 200980I110 (to J.F.L.-G.), and Medical Research Council United Kingdom Grant G0900050 (to G.M.). Recipient of a predoctoral fellowship from University of the Basque Country, Spain. Recipient of a postdoctoral fellowship from Fundacion Alicia Koplowitz, Spain. Recipient of a predoctoral fellowship from Consejo Superior de Investigaciones Científicas, Spain.

GPR39 is an orphan GPCR receptor belonging to the ghrelin/motilin receptor subfamily. The receptor is constitutively active and Zn(2+) is a physiological agonist of GPR39. The receptor is emerging as an important regulator of gastrointestinal motility and secretion. Although GPR39 does not seem to be involved in the regulation of food intake, contradictory results are available on the role of GPR39 in the regulation of body weight. A well-established stimulatory role for GPR39 has been defined in insulin secretion which makes the receptor an attractive target for the treatment of type 1 or 2 diabetes. GPR39 signaling also inhibits apoptosis and mediates neural synaptic signaling. Novel ligands of GPR39 are warranted to reveal the main physiological role of this receptor.

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

Cell-surface cytokine receptors are regulated by their cis-interacting stimulatory and inhibitory co-receptors. We previously showed that the Ig-like cell-adhesion molecule nectin-4 cis-interacts with the prolactin receptor through the extracellular region and stimulates prolactin-induced prolactin receptor activation and signaling, resulting in alveolar development in the mouse mammary gland. However, it remains unknown how this interaction stimulates these effects. We show here that the cis-interaction of the extracellular region of nectin-4 with the prolactin receptor was not sufficient for eliciting these effects and that the cytoplasmic region of nectin-4 was also required for this interaction. The cytoplasmic region of nectin-4 directly interacted with suppressor of cytokine signaling 1 (SOCS1), but not SOCS3, JAK2, or STAT5a, and inhibited the interaction of SOCS1 with JAK2, eventually resulting in the increased phosphorylation of STAT5a. The juxtamembrane region of nectin-4 interacted with the Src homology 2 domain of SOCS1. Both the interaction of nectin-4 with the extracellular region of the prolactin receptor and the interaction of SOCS1 with the cytoplasmic region of nectin-4 were required for the stimulatory effect of nectin-4 on the prolactin-induced prolactin receptor activation. The third Ig-like domain of nectin-4 and the second fibronectin type III domain of the prolactin receptor were involved in this cis-interaction, and both the extracellular and transmembrane regions of nectin-4 and the prolactin receptor were required for this direct interaction. These results indicate that nectin-4 serves as a stimulatory co-receptor for the prolactin receptor by regulating the feedback inhibition of SOCS1 in the JAK2-STAT5a signaling pathway.

International audience; Active G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients.

Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

Dopamine amides of long chain fatty acids (NADA) are a family of endogenous mammalian lipids with an unknown function; they are anti-proliferative for many cancer cell lines. The aim of this study was to identify the NADA receptor responsible for cell death induction. Using PC12 cells treated with NADA in combination with various receptor blockers, or pre-treated with the known receptor agonists to down-regulate the proposed receptor, NADA were shown to induce apoptosis. This activity was blocked only by the non-CB1-non-CB2 receptor antagonist O-1918, and by the pre-treatment with the agonists of this receptor ethanolamides of palmitic and oleic acids.

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT.

The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in Escherichia coli as a functional, ligand-binding receptor, but yields of purified receptor are still low for comprehensive structural studies. Here, several approaches have been examined to optimize the yields of active, ligand-binding receptor. Optimisation of E. coli strain and induction protocol yielded a significant improvement in expression of active receptor. Expression of the receptor in BL21(DE3) cells, in combination with autoinduction improved expression 10-fold when compared with previously reported expression protocols using IPTG-mediated induction in DH5alpha cells. Optimization of the purification protocol revealed that supplementation of buffers with phospholipids enhanced recovery of active receptor. The methods examined are potentially applicable to other GPCRs expressed in E. coli.

The effect of galanin is mediated through three GPCR subtypes, GalR1-3. The limited number of specific ligands to the galanin receptor subtypes has hindered the understanding of the individual effects of each receptor subtype. This review summarizes the current data of the importance of the galanin receptor subtypes and receptor subtype specific agonists and antagonists and their involvement in different biological and pathological functions.

Olfactory receptors, which belong to the family of G-protein-coupled receptors, are found to be ectopically expressed in non-sensory tissues mediating a variety of cellular functions. In this study we detected the olfactory receptor OR51E2 at the transcript and the protein level in human epidermal melanocytes. Stimulation of primary melanocytes with the OR51E2 ligand β-ionone significantly inhibited melanocyte proliferation. Our results further showed that β-ionone stimulates melanogenesis and dendritogenesis. Using RNA silencing and receptor antagonists, we demonstrated that OR51E2 activation elevated cytosolic Ca(2+) and cAMP, which could mediate the observed increase in melanin synthesis. Co-immunocytochemical stainings using a specific OR51E2 antibody revealed subcellular localization of the receptor in early endosomes associated with EEA-1 (early endosome antigen 1). Plasma membrane preparations showed that OR51E2 protein is present at the melanocyte cell surface. Our findings thus suggest that activation of olfactory receptor signaling by external compounds can influence melanocyte homeostasis.

G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)-binding protein)-coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems-PEN-GPR83 and bigLEN-GPR171-may be functionally coupled in the regulation of feeding.

We have previously shown in SH-SY5Y human neuroblastoma cells that the expressions of basal (75 kDa) and high molecular weight (HMW; 85 kDa) isoforms of the p75 neurotrophic receptor (p75NTR) are stimulated by amyloid-β peptide1–42 oligomers (AβOs) via the insulin-like growth factor-1 receptor (IGF-1R). On the other hand, it is known that AβOs inhibit insulin receptor (IR) signaling. The purpose of the present study was to determine the involvement of IR signaling in the regulation of p75 neurotrophin receptor (p75NTR) protein isoform expression in cultured SH-SY5Y cells and in hippocampi from late-stage human Alzheimer's disease (AD) brains. Interestingly, insulin induced the expression of basal and HMW p75NTR isoforms in SH-SY5Y cells, suggesting the presence of cross-talk between the IR and IGF-1R for the regulation of p75NTR expression. Reducing IR signaling with an IR kinase inhibitor (AG 1024) or IR-targeted siRNAs increased HMW p75NTR expression and reduced tyrosine receptor kinase-A (Trk-A) expression as well as postsynaptic density protein 95 (PSD95) expression in SH-SY5Y cells. Both basal and HMW p75NTR isoforms were increased in the hippocampi of post-mortem late-stage human AD brains (relative to non-AD brains), and the protein expression of HMW p75NTR was negatively associated with Trk-A expression, PSD95 expression, and IR expression. Thus, increased p75NTR expression, specifically an increased p75NTR-to-Trk-A ratio, is likely to play a role in synaptic loss and neuronal cell death in late-stage AD. Collectively, these findings suggest that increased expression of the p75NTR due to IR signaling inhibition by AβOs might be involved in the pathology of AD.