Search

The TCA cycle metabolite succinate functions as an intra- and extracellular signal of metabolic stress. Based on the phenotype of UCP-1-deficient mice, Mills et al. (2021) now report in Nature Metabolism that accumulation of extracellular succinate due to impaired elimination in thermogenic fat drives liver inflammation and fibrosis through the succinate receptor SUCNR1.

Background Succinate is a Krebs cycle intermediate whose formation is enhanced under metabolic stress, and for which a selective sensor GPR91 has been identified on various cell types including platelets. Platelet-derived eicosanoids play pivotal roles in platelet activation/aggregation, which is key to thrombus formation and progression of atherothrombosis. Objectives This study aims to decipher the molecular mechanism(s) and potential involvement of eicosanoids in succinate enhanced platelet activation/aggregation. Methods We used liquid chromatography-mass spectrometry (LC-MS)/MS-based lipid mediator profiling to identify eicosanoids regulated by succinate. We ran light transmittance aggregometry and flow cytometry to assess platelet aggregation, P-selectin expression, and platelet-polymorphonuclear leukocyte (PMN) adherence. Various pharmacological tools were used to assess the contributions of GPR91 signalling and eicosanoids in platelet aggregation. Results Succinate and two types of synthetic non-metabolite GPR91 agonists-cis-epoxysuccinate (cES) and Cmpd131-potentiated platelet aggregation, which was partially blocked by a selective GPR91 antagonist XT1. GPR91 activation increased production of 12-hydroxy-eicosatetraenoic acid (12-HETE), thromboxane (TX) A2 , and 12-hydroxy-heptadecatrienoic acid (12-HHT) in human platelets, associated with phosphorylation of cytosolic phospholipase A2 (cPLA2 ), suggesting increased availability of free arachidonic acid. Blocking 12-HETE and TXA2 synthesis, or antagonism of the TXA2 receptor, significantly reduced platelet aggregation enhanced by GPR91 signalling. Moreover, platelet-PMN suspensions challenged with succinate exhibited enhanced transcellular biosynthesis of leukotriene C4 (LTC4 ), a powerful proinflammatory vascular spasmogen. Conclusion Succinate signals through GPR91 to promote biosynthesis of eicosanoids, which contribute to platelet aggregation/activation and potentially vascular inflammation. Hence, GPR91 may be a suitable target for pharmacological intervention in atherothrombotic conditions.

Eicosanoids are a family of bioactive compounds derived from arachidonic acid (AA) that play pivotal roles in physiology and disease, including inflammatory conditions of multiple organ systems. The biosynthesis of eicosanoids requires a series of catalytic steps that are controlled by designated enzymes, which can be regulated by inflammatory and stress signals via transcriptional and translational mechanisms. In the past decades, evidence have emerged indicating that G-protein coupled receptors (GPCRs) can sense extracellular metabolites, and regulate inflammatory responses including eicosanoid production. This review focuses on the recent advances of metabolite GPCRs research, their role in regulation of eicosanoid biosynthesis, and the link to pathophysiological conditions.

Using single-cell RNA sequencing (RNA-seq), Kupari and coworkers (Cell Rep., 2019) have generated a long sought-after molecular atlas of vagal afferents in the mouse. Vagal afferents were found to be organized into 24 subtypes, revealing a level of diversity that was not previously recognized.

Objective: GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. Methods: Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNAseq analysis. Results: Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. Conclusions: GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells. (c) 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

GPR40 is generally known to signal through Gq. However, in transfected cells, certain synthetic agonists can make the receptor signal also through Gs and cAMP (Hauge et al., 2015). Here we find that, in colonic crypt cultures, the GLP-1 secretion induced by such Gq + Gs GPR40 agonists is indeed inhibited by blockers of both Gq and Gs and is eliminated by combining these. This in contrast to Gq-only GPR40 agonists which only are affected by the Gq inhibitor. Importantly, Gq-only GPR40 agonists in combination with low doses of selective synthetic agonists for Gs coupled receptors, e.g. GPR119 and TGR5 provide more than additive GLP-1 secretion both ex vivo and in vivo in mice. It is concluded that under physiological circumstances triglyceride metabolites, i.e. long chain fatty acids and 2-monoacyl glycerol plus bile acids, act synergistically through their respective receptors, GPR40, GPR119 and TGR5 to stimulate GLP-1 secretion robustly by combining Gq and Gs signaling pathways.

The long-chain fatty acid receptor FFAR1 is highly expressed in pancreatic β-cells. Synthetic FFAR1 agonists can be used as antidiabetic drugs to promote glucose-stimulated insulin secretion (GSIS). However, the physiological role of FFAR1 in β-cells remains poorly understood. Here we show that 20-HETE activates FFAR1 and promotes GSIS via FFAR1 with higher potency and efficacy than dietary fatty acids such as palmitic, linoleic, and α-linolenic acid. Murine and human β-cells produce 20-HETE, and the ω-hydroxylase-mediated formation and release of 20-HETE is strongly stimulated by glucose. Pharmacological inhibition of 20-HETE formation and blockade of FFAR1 in islets inhibits GSIS. In islets from type-2 diabetic humans and mice, glucose-stimulated 20-HETE formation and 20-HETE-dependent stimulation of GSIS are strongly reduced. We show that 20-HETE is an FFAR1 agonist, which functions as an autocrine positive feed-forward regulator of GSIS, and that a reduced glucose-induced 20-HETE formation contributes to inefficient GSIS in type-2 diabetes., FFAR1 receptor is highly expressed in beta cells and its activation has been suggested as therapy against type-2 diabetes. Here, Tunaru et al. show that 20-hydroxyeicosatetraenoic acid, produced within the islets upon glucose stimulation, acts in an autocrine manner to stimulate insulin secretion via FFAR1 activation.

Gut hormones send information about incoming nutrients to the rest of the body and thereby control many aspects of metabolism. The secretion of ghrelin and glucagon-like protein (GLP)-1, two hormones with opposite secretory patterns and opposite actions on multiple targets, is controlled by a limited number of G-protein coupled receptors (GPCRs); half of which recognize and bind dietary nutrient metabolites, metabolites generated by gut microbiota, and metabolites of the host's intermediary metabolism. Most metabolite GPCRs controlling ghrelin secretion are inhibitory, whereas all metabolite receptors controlling GLP-1 secretion are stimulatory. This dichotomy in metabolite sensor function, which is obtained through a combination of differential expression and cell-dependent signaling bias, offers pharmacological targets to stimulate GLP-1 and inhibit ghrelin through the same mechanism.

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and β-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) β-arrestin only; and 3) Gq and β-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways.

G-protein-coupled receptor 41 (GPR41) also called free fatty acid receptor 3 (FFAR3) is a Gαi-coupled receptor activated by short-chain fatty acids (SCFAs) mainly produced from dietary complex carbohydrate fibers in the large intestine as products of fermentation by microbiota. FFAR3 is expressed in enteroendocrine cells, but has recently also been shown to be present in sympathetic neurons of the superior cervical ganglion. The aim of this study was to investigate whether the FFAR3 is present in other autonomic and sensory ganglia possibly influencing gut physiology. Cryostat sections were cut of autonomic and sensory ganglia of a transgenic reporter mouse expressing the monomeric red fluorescent protein (mRFP) gene under the control of the FFAR3 promoter. Control for specific expression was also done by immunohistochemistry with an antibody against the reporter protein. mRFP expression was as expected found not only in neurons of the superior cervical ganglion, but also in sympathetic ganglia of the thoracic and lumbar sympathetic trunk. Further, neurons in prevertebral ganglia expressed the mRFP reporter. FFAR3-mRFP-expressing neurons were also present in both autonomic and sensory ganglia such as the vagal ganglion, the spinal dorsal root ganglion and the trigeminal ganglion. No expression was observed in the brain or spinal cord. By use of radioactive-labeled antisense DNA probes, mRNA encoding the FFAR3 was found to be present in cells of the same ganglia. Further, the expression of the FFAR3 in the ganglia of the transgenic mice was confirmed by immunohistochemistry using an antibody directed against the receptor protein, and double labeling colocalized mRFP and the FFAR3-protein in the same neurons. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) on extracts from the ganglia supported the presence mRNA encoding the FFAR3 in most of the investigated tissues. These data indicate that FFAR3 is expressed on postganglionic sympathetic and sensory neurons in both the autonomic and somatic peripheral nervous system and that SCFAs act not only through the enteroendocrine system but also directly by modifying physiological reflexes integrating the peripheral nervous system and the gastro-intestinal tract.

Objectives GPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins. Methods and results Here we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner. Conclusions It is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.

SummaryThe melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly enriched GPCR in peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent antisecretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal (i.p.) administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.

The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa.

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β(2)-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β(2)-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.

Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling.

Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.

The GPR119 receptor is expressed predominantly in pancreatic β cells and in enteroendocrine cells. It is a major target for the development of anti-diabetic drugs that through GPR119 activation may stimulate both insulin and GLP-1 release. GPR119 can be activated by oleoylethanolamide and several other endogenous lipids containing oleic acid: these include N-oleoyl-dopamine, 1-oleoyl-lysophosphatidylcholine, generated in the tissue, and 2-oleoyl glycerol generated in the gut lumen. Thus, the well-known stimulation of GLP-1 release by dietary fat is probably not only mediated by free fatty acids acting through, for example, GPR40, but is also probably mediated in large part through the luminal formation of 2-monoacylglycerol acting on the 'fat sensor' GPR119. In the pancreas GPR119 may also be stimulated by 2-monoacylglycerol generated from local turnover of pancreatic triacylglycerol. Knowledge about the endogenous physiological ligands and their mode of interaction with GPR119 will be crucial for the development of efficient second-generation modulators of this important drug target.

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6–53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe111 at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC50. In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.

From the deep part of the main ligand-binding crevice, a minor, often shallower pocket extends between the extracellular ends of transmembrane domains (TM)-I, II, III and VII of 7TM receptors. This minor binding pocket is defined by a highly conserved kink in TM-II that is induced by a proline residue located in one of two adjacent positions. Here we argue that this minor binding pocket is important for receptor activation. Functional coupling of the receptors seems to be mediated through the hydrogen bond network located between the intracellular segments of these TMs, with the allosteric interface between TM-II and TM-VII being of particular significance. Importantly, the minor binding pocket, especially the proline-kink in TM-II, is involved in G protein versus arrestin pathway-biased signaling, for example in the angiotensin AT1 system. Consequently, this pocket could be specifically targeted in the development of functionally biased drugs.

Background & Aims Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled–coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. Methods We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. Results Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline. Conclusions In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation.

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Gαi as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response element-driven transcriptional activity in a pertussis toxin-sensitive manner. Gαs and Gαq were not activated constitutively as determined by the lack of cAMP production, the lack of inositol phosphate turnover, and the lack of activities of the transcription factors: cAMP response element-binding protein and nuclear factor-κB. Immunohistochemistry and confocal microscopy of FLAG- and green fluorescent protein-tagged EBI2 revealed cell-surface expression. A putative N-terminal truncated version of EBI2, Δ4-EBI2, showed similar expression and signaling through Gαi as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based on the constitutive signaling and cellular expression pattern of EBI2, it is suggested that it may function in conjunction with BILF1 in the reprogramming of the cell during EBV infection.

The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC50: 1-10 nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs. ispartof: Biochemical Pharmacology vol:70 issue:5 pages:752-761 ispartof: location:England status: published

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the β2-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the δ-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor.

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G(q), phospholipase C pathway was approximately 50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G(12/13) and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Gαi, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.

Ghrelin plays a key role as the major orexigenic hormone from the gastrointestinal tract to the hypothalamic areas that govern food intake, balancing against a multitude of anorectic hormones, such as leptin, insulin and PYY 3–36 . Surprisingly, even in the absence of agonist, the ghrelin receptor signals with ∼50% activity. Thus, although ghrelin receptor antagonists are expected to reduce meal-associated food intake, inverse agonists of the ghrelin receptor, by blocking the constitutive receptor activity, might lower the set-point for hunger between meals, eliminating the craving for second orders, desserts and snacks.

AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp171 (AspIV:20), Asp262 (AspVI:23), and Glu288 (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp171 in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp262 and Glu288 from TM-VI and -VII, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp262 and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral bioavailability can be designed to mimic this interaction and thereby efficiently and selectively block the CXCR4 receptor.

The chemokine-like, secreted protein product of the U83 gene from human herpesvirus 6, here named vCCL4, was chemically synthesized to be characterized in a complete library of the 18 known human chemokine receptors expressed individually in stably transfected cell lines. vCCL4 was found to cause calcium mobilization as efficiently as the endogenous chemokine ligand CCL2 through the CCR2 receptor, whereas the virally encoded chemokine did not affect any of the other 17 human chemokine receptors tested. Mutual cross-desensitization between CCL2 and vCCL4 was demonstrated in the CCR2-transfected cells. The affinity of vCCL4 for the CCR2 receptor was 79 nm as determined in competition binding against radioactively labeled CCL2. In the murine pre-B lymphocyte cell line L1.2 stably transfected with the CCR2 receptor, vCCL4 acted as a relatively low potency but highly efficacious chemoattractant being equally or more efficacious in causing cell migration than CCL2 and CCL7 and considerably more efficacious than CCL8 and CCL13. It is concluded that human herpesvirus 6 encodes a highly selective and efficacious CCR2 agonist, which will attract CCR2 expressing cells, for example macrophages and monocytes, conceivably for the virus to infect and to establish latency in. It is suggested that vCCL4 during reactivation of the virus in for example monocyte-derived microglia could perhaps be involved in the pathogenesis of the CCR2-dependent disease, multiple sclerosis.

Summary Peptide YY (PYY) is released following food intake and regulates intestinal function and glucose homeostasis, but the mechanisms underpinning these processes are unclear. Enteroendocrine L cells contain PYY and express the acylethanolamine receptor, Gpr119. Here, we show that Gpr119 activation inhibited epithelial electrolyte secretion in human and mouse colon in a glucose-sensitive manner. Endogenous PYY selectively mediated these effects, since PYY−/− mice showed no Gpr119 response, but responses were observed in NPY−/− mice. Importantly, Gpr119 responses in wild-type (WT) mouse tissue and human colon were abolished by Y1 receptor antagonism, but were not enhanced by dipeptidylpeptidase IV blockade, indicating that PYY processing to PYY(3-36) was not important. In addition, Gpr119 agonism reduced glycemic excursions after oral glucose delivery to WT mice but not PYY−/− mice. Taken together, these data demonstrate a previously unrecognized role of PYY in mediating intestinal Gpr119 activity and an associated function in controlling glucose tolerance., Highlights ► Endogenous PYY, but not NPY, mediates Gpr119 effects in human and mouse colon mucosa ► The action of endogenous PYY is mediated specifically via epithelial Y1 receptors ► Apical and basolateral Gpr119 responses are glucose sensitive ► Gpr119 agonism reduced glycemia after oral glucose in WT but not PYY−/− mice

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

Enteroendocrine cells, which secrete peptide hormones in response to sensation of food and gut microbiota products, can now be genetically tagged, isolated, cultured, and characterized for expression of the elusive chemosensors, as shown in publications in PNAS (Samuel et al., 2008) and in this issue (Reimann et al., 2008).

As the temperature and humidity rose in the auditorium during the heat-wave of the Italian alps, it became evident that, in fact, more was revealed of the bodies of certain distinguished pharmacologists than of the true nature of Stephenson's efficacy.Nevertheless, real 3D structures of receptors in their empty, apo-, form as well as in their holo-forms, i.e. in complex with different ligands: agonists, partial agonists, antagonists, etc. are currently becoming available, at least in the nuclear receptor field (Fig. 2Fig. 2). Thus, we are rapidly approaching a long-awaited point in time where pharmacologists and medicinal chemists can relate theoretical pharmacological concepts directly to 3D structures. This could soon be the case even for 7TM receptors. On the day of the meeting the structural basis for activation of the G protein, Ras, by the nucleotide exchange factor, Sos, was published[10xBorlack-Sjodin, P.A., Margerit, S.M., Bar-Sagi, D., and Kuriyan, J. Nature. 1998; 394: 337–343Crossref | PubMed | Scopus (430)See all References[10]. The `loosening' of the GDP (corresponding to efficacy—see above) was clearly shown to be achieved through two antiparallel helices extending from the Sos molecule and interacting with a switch domain on the G protein. It could be relevant that the main G protein-interacting domain of 7TM receptors also is represented by two antiparallel helices, i.e. the intracellular extensions of TM5 and TM6 (Ref. [11xAltenbach, C. et al. Biochemistry. 1995; 35: 12470–12478Crossref | Scopus (263)See all References[11]; Fig. 1Fig. 1).

If two ligands bind to the same receptor, but do not readily compete with each other in radioligand- binding assays, the most straightforward explanation is that they bind to distinct sites on the receptor (A in Fig. 3Fig. 3). However, substance P (SP) and neurokinin A (NKA) are structurally homologous, and mutations in the NK1 receptor that have been shown to affect SP binding also affect the binding of NKA and neurokinin B (NKB) to this receptor (Fig. 1Fig. 1)[31xFong, T.M., Huang, G.G., and Strader, C.D. J. Biol. Chem. 1992; 267: 25664–25667PubMedSee all References, 32xStrader, C.D., Fong, T.M., Tota, M.R., Underwood, D., and Dixon, R.A. Annu. Rev. Biochem. 1994; 63: 101–132Crossref | PubMedSee all References]. Moreover, although NKA and the other septide-like ligands compete poorly with SP for binding to the NK1 receptor, SP competes with very high affinity for binding with radiolabelled NKA ([33xHastrup, H. and Schwartz, T.W. FEBS Lett. 1996; 399: 264–266Abstract | Full Text PDF | PubMed | Scopus (88)See all References[33]).Fig. 3Two models to explain how substance P (SP) and septide-like ligands might bind to the same receptor, but compete poorly in radioligand-binding assays. Ins (1, 4, 5)P3, inositol (1, 4, 5) trisphosphate; G, G protein; NKA, neurokinin A; NKB, neurokinin B.View Large Image | Download PowerPoint SlideAnother possibility (B) is that the NK1 receptor has two active conformers: (1) a general-tachykinin conformer (denoted R*gen), which binds the majority of tachykinin peptides (NKA, NKB, septide and possibly also SP) with equal, relatively high affinities (0.5 nm) and which activates the phospholipase C (PLC) pathway rather selectively; and (2) an SP-preferring conformer (denoted R*SP), which binds SP with very high affinity (0.05 nm) and the other septide-like ligands with much lower affinities (∼50 nm) and which can activate both the PLC pathway and the adenylate cyclase pathway. The actual binding sites could be variations of the same site presented with some crucial structural difference by the two conformers. Radiolabelled SP at low concentrations preferentially binds to the SP-preferring conformer. Importantly, as the septide-like ligands compete poorly with SP binding, the interconversion from the SP-preferring conformer to the general-tachykinin conformer does not occur readily. The structural or cell-biological basis for these putative distinct conformers of the NK1 receptor is not clear. However, they could represent distinct complexes between the receptor and different G proteins or G protein subunits. The interaction between receptors and G proteins in a cell is known to be far from random and, for example, βγ subunits are basically immobilized through interaction with the cytoskeleton[34xNeubig, R.R. FASEB J. 1994; 8: 939–946PubMedSee all References[34]. Another possibility could be that some ligands and G protein subunits preferentially bind to either monomeric or dimeric forms of the receptor[35xHerbert, T.E. et al. J. Biol. Chem. 1996; 271: 16384–16392Crossref | PubMed | Scopus (597)See all References[35], which might not interconvert readily. As SP competes readily and with very high affinity for NKA and septide binding[33xHastrup, H. and Schwartz, T.W. FEBS Lett. 1996; 399: 264–266Abstract | Full Text PDF | PubMed | Scopus (88)See all References[33], then the interconversion from the putative, general-tachykinin- binding conformer to the SP-preferring conformer must occur readily.The phenomenon of an agonist being a high-affinity binder and receptor activator, but lacking the ability to compete for binding against other ligands, was initially demonstrated in mutant NK1 receptors with substitutions in transmembrane segment II ([36xRosenkilde, M.M., Cahir, M., Gether, U., Hjorth, S.A., and Schwartz, T.W. J. Biol. Chem. 1994; 269: 28160–28164PubMedSee all References[36]). Here, SP had lost the ability to compete for binding with a radiolabelled nonpeptide antagonist, but still bound with high affinity in homologous binding assays[36xRosenkilde, M.M., Cahir, M., Gether, U., Hjorth, S.A., and Schwartz, T.W. J. Biol. Chem. 1994; 269: 28160–28164PubMedSee all References[36]. Similar observations have been made in mutant κ-opioid receptors for both peptide and nonpeptide agonists against radiolabeled nonpeptide antagonists[37xHjorth, S.A., Thirstrup, K., and Schwartz, T.W. Mol. Pharmacol. 1996; 50: 977–984PubMedSee all References[37]. However, a similar phenomenon has also been described in non-mutated wild-type receptors. Thus, it was recently found in the newly identified NK3B receptor that the IC50 for NKB in competition with radiolabelled senktide (another tachykinin agonist) was >10 μm, although NKB at this receptor stimulated phosphatidylinositol turnover with an EC50 of 1 nm ([38xKrause, J.E. et al. Proc. Natl. Acad. Sci. U. S. A. 1996; 94: 310–315Crossref | Scopus (38)See all References[38]). In analogy with NKA and septide at the NK1 receptor, NKB must bind to the NK3B receptor with a nanomolar affinity, but cannot compete for binding with radiolabelled senktide. Similarly, in the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, the homologous peptide, vasoactive intestinal polypeptide (VIP), competes for binding against radioactive PACAP with a micromolar affinity; nevertheless, in homologous binding experiments performed with the same receptor, VIP was recently shown to have a nanomolar affinity[39xHashimoto, H. et al. Mol. Pharmacol. 1997; 51: 128–135See all References[39]. It is likely that many more of these stories will be disclosed when homologous binding experiments with agonists become more widely used.A surprising apparent increase in affinity for septide-like ligands, including NKA and NKB, has been observed in some mutant NK1 receptors with point substitutions in transmembrane segments II, IV and V, as well as in some chimeric constructs between the NK1 and NK3 receptors[40xHuang, R.R., Huang, D., Strader, C.D., and Fong, T.M. Biochemistry. 1995; 34: 16467–16472Crossref | PubMed | Scopus (34)See all References, 41xTian, Y., Wu, L-H., Oxender, D.L., and Chung, F-Z. J. Biol. Chem. 1996; 271: 20250–20257Crossref | PubMed | Scopus (23)See all References, 42xRiitano, D., Werge, T.M., and Costa, T. J. Biol. Chem. 1997; 272: 7646–7655Crossref | PubMed | Scopus (19)See all References]. As these ligands already bind with high affinity to the wild-type NK1 receptor[33xHastrup, H. and Schwartz, T.W. FEBS Lett. 1996; 399: 264–266Abstract | Full Text PDF | PubMed | Scopus (88)See all References[33], it could be argued that the mutations enhance the ability of the septide ligands to compete for binding with radiolabelled SP, rather than increase the affinity for the ligands. It should be noted that the experiments showing an apparent increase in on-rate for septide-like ligands caused by one of these mutations was actually performed only indirectly, that is, in competition with radiolabelled SP ([40xHuang, R.R., Huang, D., Strader, C.D., and Fong, T.M. Biochemistry. 1995; 34: 16467–16472Crossref | PubMed | Scopus (34)See all References[40]).Mutations that affect the allosteric transition between conformations that each bind ligands with high affinity, instead of affecting the actual affinity of the ligands[36xRosenkilde, M.M., Cahir, M., Gether, U., Hjorth, S.A., and Schwartz, T.W. J. Biol. Chem. 1994; 269: 28160–28164PubMedSee all References[36], have also been described recently in, for example, the enzyme thrombin[43xGibbs, C.S. et al. Nature. 1995; 378: 413–416Crossref | PubMedSee all References[43] and the nicotinic acetylcholine receptor[44xGalzi, J-L., Edelstein, S.J., and Changeux, J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1853–1858Crossref | PubMed | Scopus (80)See all References[44].

The ghrelin receptor (GhR) is a class A G protein-coupled receptor (GPCR) involved in the entero-endocrine signaling systems that regulates food intake and energy homeostasis. The GhR is noted for its unusually high basal constitutively activity. GhR is a potential drug target for “diabesity” syndromes, and the interaction between GhR and its endogenous peptide ligand, ghrelin, has been intensively studied. However, there is only a limited understanding of GhR pharmacology and its molecular mechanism of signal transduction. Using well-established amber codon suppression technology and state-of-the-art single-molecule techniques, we are developing tools to monitor directly differential conformational dynamics of GhR in the presence and absence of its binding partners, including ligands, G proteins, or other GPCRs. For example, we are preparing single-site and double-site fluorescently labeled GhR and a series of labeled ghrelin analogues. These engineered receptors can be studied in cell-based systems or reconstituted in NABBs (Nanoscale Apolipoprotein Bound Bilayers) after purification. These types of approaches will enable us to better understand the complexity of GhR signaling in the neuro-endocrine system, providing insights to design specific drugs for targeting fine-tuned signal pathways involved in metabolic disorders like obesity and diabetes.

Non-peptide ligands for peptide receptors for the G-protein-coupled type are generally antagonists, except in the opiate system. Recently, it was observed that a subset of biphenylimidazole derivatives surprisingly possessed angiotensin-like activity in vivo. In COS-7 cells transfected with the rat AT1 receptor a prototype of these compounds, L-162,313 stimulated phosphoinositide hydrolysis with an EC50 of 33 +/- 11 nM. The maximal response to the compound was 50% of that of angiotensin II in COS-7 cells but only 3% in stably transfected Chinese hamster ovary cells. The agonistic effect of L-162,313 was blocked by the AT1-specific antagonist L-158,809 and was not observed in untransfected cells. In Chinese hamster ovary cells, L-162,313 also acted as an insurmountable antagonist of the angiotensin stimulated phosphoinositide hydrolysis. In contrast to previously tested non-peptide ligands, L-162,313 bound with reasonably high affinity to the Xenopus laevis AT1 receptor. In the human receptor, the binding of L-162,313 was found to be unaffected by point mutations in transmembrane segments III and VII, which impaired the binding of biphenylimidazole antagonists. Substitutions in the extracellular domains of the human and rat receptor, which impaired the binding of angiotensin II, did not affect the binding of L-162,313. It is concluded that a subset of biphenylimidazole compounds can act as high affinity partial agonists on the AT1 receptor. These compounds have molecular interactions with the receptor which appear to differ both from that of the structurally similar non-peptide antagonists and from that of their functional counterpart, the peptide agonist.

Glucagon and glucagon-like peptide 1 (GLP-1) are homologous peptide hormones that are recognized by likewise homologous, but highly selective receptors. Analogs of glucagon and GLP-1, in which the divergent residues were systematically exchanged, were employed to identify the structural requirements for their selective receptor recognition. Substitutions in the NH2-terminal part of the glucagon molecule with the corresponding GLP-1 residues, as for example in [Ala2,Glu3]-glucagon and [Val10,Ser12]glucagon, reduced the binding affinity for the glucagon receptor several hundred-fold without increasing the affinity for the GLP-1 receptor. In contrast, introduction of GLP-1 residues into the far COOH-terminal part of the glucagon molecule, e.g. [Val27,Lys28,Gly29,Arg30]glucagon, had a minimal effect on recognition of the glucagon receptor, but improved the affinity of the analog for the GLP-1 receptor up to 200-fold. Similarly, substitutions in especially the far COOH-terminal part of the GLP-1 molecule with the corresponding glucagon residues, e.g. des-Arg30-[Met27,Asn28,Thr29]GLP-1, decreased the affinity for the GLP-1 receptor several hundred-fold (IC50 = 0.4-190 nM) without increasing the affinity for the glucagon receptor. Conversely, substitutions in the NH2-terminal part of the GLP-1 molecule impaired the affinity for the GLP-1 receptor only moderately. We conclude that the selective recognition of the glucagon and GLP-1 receptors is determined by residues located at opposite ends of the homologous peptide ligands. This conclusion is supported by the observation that a "chimeric" peptide consisting of the NH2-terminal part of the glucagon molecule joined to the COOH-terminal part of the GLP-1 molecule was recognized with high affinity by both receptors.

To locate essential determinants for angiotensin II binding, we have performed a systematic mutational analysis of the exterior domain of the AT1 receptor. Receptor mutants, deficient in peptide binding, were analyzed using radiolabeled nonpeptide ligand as an important tool. Two independent strategies for mutagenesis were employed: conservative segment exchange and point mutagenesis of evolutionarily conserved residues. Results from the conservative segment exchange in which 6-17 residues were replaced with chemically similar, yet different, amino acid sequences of the same length suggested that important peptide ligand binding epitopes are located in the N-terminal extension of the AT1 receptor, in particular adjacent to the top of transmembrane segment I (TM-I), and in the third extracellular loop, close to the top of TM-VII. The substitution of residues from either of these regions resulted in a 5,000-20,000-fold decrease in affinity for the peptide agonist angiotensin II (AII) and the peptide antagonist [Sar1,Leu8]AII without affecting the binding of nonpeptide antagonists. Alanine substitution of evolutionarily conserved residues demonstrated that peptide binding was dependent on several residues in the N-terminal extension, near the top of TM-I, a tyrosine residue located in extracellular loop 1, close to TM-II, and 2 aspartate residues positioned in extracellular loop 3 on the same face of an alpha-helical extension of TM-VII. In all cases the binding of nonpeptide antagonist was unaffected by these substitutions. It is concluded that important epitopes involved in angiotensin II binding are located around the top of transmembrane segments I, II, and VII which conceivably are in close spatial proximity in the folded receptor structure.

Mutational analysis of the NK-1 receptor indicates that residues involved in non-peptide antagonist binding cluster around the outer portion of transmembrane segments (TM) V and VI. In contrast mutations affecting the binding of the natural peptide agonist, substance P, are scattered in the exterior part of the receptor. Recently it was reported that a number of mutations in TM-II also seriously impair substance P binding. Here we confirm that Ala substitutions for these residues located on a hydrophilic helical face of TM-II basically eliminate substance P binding to the NK-1 receptor, provided that a radiolabeled non-peptide antagonist is used as radioligand. Surprisingly, radiolabeled substance P bound well to all these mutant receptors and was displaced with only slightly reduced affinity by the unlabeled peptide and by the non-peptide antagonists. The wild-type homologous NK-2 receptor displayed properties similar to those observed in the mutated NK-1 receptors, i.e. concomitant high affinity binding of radiolabeled agonist peptide (in this case neurokinin A), yet low affinity, G-protein independent competition of unlabeled peptide with radiolabeled non-peptide antagonist. It is concluded that substitutions in TM-II of the NK-1 receptor do not affect the high affinity binding of substance P but instead block the ability of the peptides to compete for non-peptide antagonist binding. It is suggested that certain mutations can impair interchange between receptor conformations that each bind different ligands with high affinity.

Previously we have found that binding of the nonpeptide substance P antagonist, CP 96,345, to the neurokinin-1 (NK-1) receptor was critically dependent on two short segments adjacent to the top of transmembrane segments (TM) V and VI, called segments A (residues 183-195) and D (residues 271-276), respectively. In the present study we have systematically performed substitutions of nonconserved residues within these two segments with residues from the homologous NK-3 and/or NK-2 receptor. In segment A, deletion of residues Glu193 and Lys194, which are not present in the NK-3 receptor, or substituting them with leucines as in the NK-2 receptor, decreased the affinity of CP 96,345 10- and 22-fold, respectively. Surprisingly, switching the position of Glu193 and Lys194 did not affect the affinity of CP 96,345, suggesting that, rather than interacting directly with CP 96,345, an interaction of these residues with one another is important for CP 96,345 binding. In segment D substitution of Tyr272 with threonine as in the NK-2 receptor and with alanine as in the NK-3 receptor decreased the affinity of CP 96,345 7- and 24-fold, respectively. Mutation of the preceding Pro271 to glycine alone did not affect CP 96,345 binding, but, combined with the mutation of Tyr272 to threonine, the affinity decreased 28-fold. A series of CP 96,345 analogues with modifications of the major chemical moieties exhibited equally reduced affinity as that of CP 96,345 for the Tyr272- and Lys193-Glu194-substituted constructs, except CP 95,555, which lacks one of the phenyl rings in the benzhydryl group and which was almost unaffected by these mutations. In conclusion, our data indicate a direct interaction between CP 96,345 and Tyr272, which are located at the top of TM VI likely in close spatial proximity to the previously identified interaction point, His197, at the top of the adjacent TM V. Furthermore, the data demonstrated a critical involvement in CP 96,345 binding of Lys193 and Glu194 located one alpha-helical turn above His197.

The processing of two homologous precursors, pro-neuropeptide Y (pro-NPY) and pro-pancreatic poly-peptide (pro-PP), was studied in four neuroendocrine cell lines after transfection: CA-77 medullary thyroid carcinoma cells, AtT-20 corticotrope pituitary cells, RIN2A-19 pancreatic endocrine cells, and NB1 neuroblastoma cells. Northern blot analysis indicated that the AtT-20 cells only expressed precursor convertase 3; in contrast, NB1 cells only expressed precursor convertase 2, whereas the RIN2A-19 and CA-77 cells expressed both enzymes. Despite these differences in expression pattern of precursor convertases the four cell lines were, surprisingly, indistinguishable in respect to their processing of pro-PP and pro-NPY. In all four cell lines, pro-NPY was almost completely converted to NPY, and, in all four cell lines, only around 50% of the PP precursor was converted to PP. The relatively poor processing efficiency of pro-PP was rather similar to the processing efficiency of the endogenously produced precursors in the respective cell lines, pro-calcitonin (CA-77), proopiomelanocortin (AtT-20), proinsulin (RIN2A-19), and pro-vasoactive intestinal polypeptide (NB1). At least in the CA-77 cells, NPY and PP were apparently sorted to the regulated secretory pathway, as upon stimulation with secretagogue the release of the transfected peptides increased in parallel with the endogenously expressed peptide, calcitonin gene-related peptide. Mutagenesis studies showed that on the N-terminal side of the di-basic processing site, the otherwise important difference in structure between PP and NPY, a proline for glutamine in position 34, was not responsible for the difference in processing efficiency. On the C-terminal side of the processing site, the efficient processing of pro-NPY could not be transferred to pro-PP by exchanging the whole C-terminal domains of the precursors. It is concluded that pro-NPY is processed more efficiently than pro-PP in all neuroendocrine cell lines tested independent on their expression of the two main precursor convertases and that mutagenesis data indicate that the structural element responsible for the efficient processing of pro-NPY is not located on the N-terminal side of the dibasic processing site.

The NK1 (substance P) and NK3 (neurokinin B) receptors are G protein-coupled receptors sharing approximately 70% identity within the membrane-spanning domains. However, they each have a distinct pharmacological profile in respect of peptide binding. To identify epitopes that determine their selectivity for natural and synthetic tachykinin peptides, we constructed a series of chimeric NK1/NK3 receptors in which carboxyl-terminal segments of increasing length in the NK1 receptor were exchanged with the corresponding segments from the NK3 receptor. The general, structural integrity of the chimeric constructs was confirmed by the amphibian tachykinin peptide, eledoisin, which was recognized equally well by both of the wild-type receptors and bound with a similar or even higher affinity to all the chimeric receptors. Competition binding studies showed that the affinity of the two natural ligands, substance P and neurokinin B, changed gradually through the series of chimeric receptors indicating that several binding epitopes throughout the receptor structure are involved in the selective recognition of these peptides. However, whereas the single, largest change in binding affinity for substance P occurred when segments in the amino-terminal end of the receptor were exchanged, this occurred for neurokinin B in the carboxyl-terminal end of the receptor. The affinity of the NK1-selective ligand, [Sar9,Met(O2)11]SP, changed even more gradually through the series of chimeric receptors than that of substance P. In contrast, the NK3-selective hexapeptide, senktide, was recognized only when transmembrane segment III and IV from the NK3 receptor were incorporated into the chimeric constructs. These data suggest that several receptor domains contribute to the binding specificity of tachykinin agonists but in varying degrees for each peptide. It is concluded that the tachykinin peptides, in partially different ways, interact with multiple epitopes scattered throughout the receptor structure, but conceivably these epitopes are closely located in space around a hypothetical receptor center.

The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic expression vectors encoding either cystatin C, KDEL extended cystatin C, or cystatin C extended with a control sequence. It is concluded that cystatin C with the KDEL tetrapeptide as a C-terminal extension is retained intracellularly without apparent accumulation of the molecule.

The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with paptide binding experiments in Y1 and PP receptor assays on rat PC-12 cells and Y2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragments of NPY, NPY-(19–36) and NPY-(11–36), indicated that NPY acts through a Y2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile31,Gln34]PP and [Leu31,Pro33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y2 and PP receptor assays. [Ile31,Gln34]PP, which bound to the Y2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.

Neuropeptide Y (NPY) belongs to the pancreatic polypeptide fold (PP-fold) family of regulatory peptides. Analysis of circular dicroic spectra of NPY showed that it has a high degree of secondary structure in aqueous solution which is in agreement with the globular, folded crystal structure of PP. Using three different approaches with synthetic peptides, we have probed the importance of the PP-fold structure in the interaction of NPY with two types of binding sites, YI and YZ receptors. First, stepwise construction of the NPY molecule from the C-terminal amidated end, showed that although C-terminal fragments encompassing most of the long a-helix reacted reasonably well with the Y2 receptor, both Y, and YZ receptors required the presence of both ends of the PP-fold for full activity. Second, perturbation of the PP-fold by substitution with a helix-breaking proline residue, resulted in the loss of recognition of the N-terminal segment of the molecule by both types of receptors. Finally, a hybrid analog was constructed in which the essential, but by itself inactive, C-terminal segment of NPY was joined with the PP-fold motif of PP. This segment of PP is only 43% homologous to the similar motif in NPY, and most of the common residues cluster in the hydrophobic core of the fold. Nevertheless, the hybrid analog reacted with almost full potency on the Yz receptors. It is concluded that the antiparallel PPfold is of structural importance for the receptor binding of NPY, and that its main function is to present the combined C- and N-terminal segments of the molecule to the receptors. Neuropeptide Y (NPY)’ is an important regulator of neuronal function with a widespread distribution. In the central nervous system, NPY is involved mainly in the regulation of food intake, memory processing, and circadian rhythm. In the peripheral nervous system, NPY functions especially as a cotransmitter with norepinephine in the regulation of vascular * The work was supported in part by the Biotechnology Centre for Neuropeptide Research, the NOVO, the Carlsberg, and the Vissing Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked