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ecent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision.

Homeostasis of the human immune system is regulated by many cellular components, including two neuropeptides, VIP and PACAP, primary stimuli for three class B G protein-coupled receptors, VPAC1, VPAC2, and PAC1. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) regulate intestinal motility and secretion and influence the functioning of the endocrine and immune systems. Inhibition of VIP and PACAP receptors is an emerging concept for new pharmacotherapies for chronic inflammation and cancer, while activation of their receptors provides neuroprotection. A small number of known active compounds for these receptors still impose limitations on their use in therapeutics. Recent cryo-EM structures of VPAC1 and PAC1 receptors in their agonist-bound active state have provided insights regarding their mechanism of activation. Here, we describe major molecular switches of VPAC1, VPAC2, and PAC1 that may act as triggers for receptor activation and compare them with similar non-covalent interactions changing upon activation that were observed for other GPCRs. Interhelical interactions in VIP and PACAP receptors that are important for agonist binding and/or activation provide a molecular basis for the design of novel selective drugs demonstrating anti-inflammatory, anti-cancer, and neuroprotective effects. The impact of genetic variants of VIP, PACAP, and their receptors on signalling mediated by endogenous agonists is also described. This sequence diversity resulting from gene splicing has a significant impact on agonist selectivity and potency as well as on the signalling properties of VIP and PACAP receptors., SCOPUS: re.j, info:eu-repo/semantics/published

Pattern separation is the process of transforming highly similar sensory inputs into distinct, dissimilar representations. It takes place in the hippocampus and is thought to be used in episodic memory. Impaired pattern separation performance has been recognized as a predictor for the development of cognitive impairments such as dementia in humans and as being present in patients with schizophrenia and post-traumatic stress disorder (PTSD). In this protocol, we describe how to implement a simple and robust object pattern separation (OPS) task in mice and rats that we have previously established and validated. This two-trial memory task uses specific object locations so differences in performance can be calibrated with the extent of object movement. Changes in performance are indicative of spatial pattern separation. In contrast to other pattern separation tasks, the OPS task allows detection of spatial pattern separation performance bidirectionally. Furthermore, the OPS task is cheaper and easier to use and interpret than other tasks that use more than two objects or that are touch-screen based. The entire protocol, from vivarium acclimatization to training of the animals, takes similar to 35-41 d. After successful training, the animals can be tested repeatedly, and three OPS experiments (n = 20-24 per experimental day) can be performed per week. A standard level of expertise in behavioral studies in rodents is sufficient to successfully integrate this paradigm into an existing rodent test battery.

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development. © 2018 National Academy of Sciences. All rights reserved.

We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.

Chemokines and their cell surface G protein-coupled receptors are critical for cell migration, not only in many fundamental biological processes but also in inflammatory diseases and cancer. Recent X-ray structures of two chemokines complexed with full-length receptors provided unprecedented insight into the atomic details of chemokine recognition and receptor activation, and computational modeling informed by new experiments leverages these insights to gain understanding of many more receptor:chemokine pairs. In parallel, chemokine receptor structures with small molecules reveal the complicated and diverse structural foundations of small molecule antagonism and allostery, highlight the inherent physicochemical challenges of receptor:chemokine interfaces, and suggest novel epitopes that can be exploited to overcome these challenges. The structures and models promote unique understanding of chemokine receptor biology, including the interpretation of two decades of experimental studies, and will undoubtedly assist future drug discovery endeavors.

Das aus dem Apoprotein Opsin und dem kovalent gebundenen Liganden bestehende Rhodopsin dient als Modellsystem für den Aktivierungsmechanismus der größten Klasse von G-Protein-gekoppelten Rezeptoren (GPCR). Infolge einer photochemischen Reaktion vollführt Rhodopsin eine Bewegungsabfolge von Sekundärstrukturelementen, wodurch es aktiviert wird, das G-Protein bindet und den Stimulus auf zellinterne Signalwege überträgt. Mithilfe der ortsspezifischen Mutagenese wurden Mutanten des bovinen Rhodopsins erzeugt, in eine künstliche Lipidumgebung eingelagert und hauptsächlich mittels FTIR-Spektroskopie untersucht. Anhand der Y191F- und Y192F-Mutanten konnte die Translokation des transienten Gegenions der Schiffschen Base Glu181 während der Aktivierung bestimmt werden. Die Interaktionen des Tyr206 sind für die gekoppelte Bewegung von EL2 und TM5 mitbestimmend, was mittels Y206F-Mutante gezeigt wurde. Eine Anhäufung von Methioninen auf der cytoplasmatischen Seite des Rezeptors ist u.a. für das Ausklappen der TM6 zuständig. Diese Bewegung ist wichtige Determinante der Rezeptoraktivierung. Hierfür wurden insgesamt fünf Mutanten verwendet. Im zweiten, hauptsächlichen Teil der Arbeit wird das bislang kaum untersuchte humane Rhodopsin mit dem bovinen Rezeptor verglichen. Ausgehend von verschiedenen Dunkelzuständen, konnte gezeigt werden, dass die Aktivierungsmechanismen beider Rezeptoren voneinander divergieren, um letztlich bei der Bildung der aktiven Spezies wieder zu konvergieren. Über die Analyse der Aminosäuresequenzen der Mammalia-Rhodopsine wurden zwei Bereiche hoher Variabilität identifiziert, die u.a. die molekulare Ursache für diese Diskrepanzen liefern. Diese Feststellung wurde mit human-bovinen-Rhodopsinchimären bewiesen. Ergänzend zu dieser Studie wurde Schafsrhodopsin einem Vergleich sowohl mit bovinem als auch mit humanem Rezeptor unterzogen. Es zeigte, als eine weitere natürlich vorkommende Variante des Lichtrezeptors, einen eigenständigen Weg der Aktivierung., Rhodopsin, which consists of the apoprotein opsin and its covalently bound ligand, is used as a model system to understand the activation mechanism of the large family of G protein coupled receptors (GPCRs). As a result of a photochemical reaction, rhodopsin undergoes activating structural changes, enabling it to bind the G protein and transmitting the stimulus to intracellular signaling pathways. In the first part of this work, site-directed mutants of bovine rhodopsin were produced, incorporated into an artificial lipid environment, and studied mainly by FTIR spectroscopy. The translocation of the transient Schiff base counterion (Glu181) during the activation process was determined using the Y191F- and Y192F-mutants. The interactions of Tyr206 contributed to the coupled movement of EL2 and TM5, which was shown by Y206F-mutant. A striking accumulation of methionines on the cytoplasmic side of the receptor was observed to be a key-player for the activating outward motion of TM6. In the second and primary part of this work, human rhodopsin, which has been rarely studied, was compared with the bovine receptor. Starting from various dark states, it was shown that the activation mechanisms of both receptors diverge from each other and yet ultimately converge in the formation of the active species. By analyzing the amino acid sequences of mammalian rhodopsins, two regions of high variability were identified, which provide the molecular basis for these discrepancies. This finding was verified by the investigation of human/bovine rhodopsin chimeras. In addition to this study, ovine rhodopsin was compared with both the bovine and human forms. It showed, as another naturally occurring variant of the light receptor, an independent pathway of activation.

Coronaviruses from both the Alpha and Betacoronavirus genera, interfere with the type I interferon (IFN) response in various ways, ensuring limited activation of the IFN response in most cell types. Of Gammacoronaviruses that mainly infect birds, little is known about activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells and in a chicken cell line. Ifnß expression in fact, is delayed with respect to the peak of viral replication and accompanying accumulation of dsRNA. In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the IFN response at the transcriptional and translational level. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, signalling of non-self dsRNA through both MDA5 and TLR3 remains intact in IBV-infected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses.

Relaxin is a peptide hormone which plays a role in mediating a wide variety of physiologies; specifically its positive cardiovascular effects have led to its success in phase three clinical trials for the treatment of acute heart failure. With this therapeutic potential, design of smaller relaxin analogues with oral bioavailability would be beneficial. To achieve this, a sound understanding of the molecular mechanisms of relaxin mediated activation of its G protein-coupled receptor (GPCR) relaxin family peptide receptor 1 (RXFP1) is required. RXFP1 is a class A, rhodopsin like GPCR which possesses a large N-terminal domain consisting of a series of 10 leucine rich repeats and is consequently classified as a leucine rich repeat (LRR) containing GPCR (LGR). Unique to other LGRs and other GPCRs, RXFP1 contains an N-terminal low density lipoprotein class A module (LDLa). Relaxin mediated activation of RXFP1 requires multiple interactions; a well characterised high-affinity interaction at the LRRs positions relaxin for a poorly characterised, lower-affinity interaction within the extracellular loops (ELs) of the transmembrane domain. Additionally, the LDLa module is absolutely required for receptor activation. The current model proposes that RXFP1 exists at the cell surface as a dimer. Relaxin binding the LRRs and ELs of one receptor in the dimer positions the N-terminal LDLa module of the relaxin bound receptor at the ELs of the other receptor in the dimer. Molecular characterisation of interactions at the ELs has been limited due to the inherent difficulties in studying membrane associated GPCRs. This project aimed to confirm interactions between relaxin / LDLa module and the ELs of RXFP1 and then characterise these interactions using NMR. A tool for investigating the interactions at the ELs in isolation of the whole receptor was designed; a soluble protein scaffold system displaying EL1 and EL2 from RXFP1 which were inserted into the native loops of a thermostabilised

Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in E. coli. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in E. coli as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.

Since many of newly synthesised D-secoestratriene derivatives showed antiestrogenic effect, with almost a total loss of estrogenic activity, we studied the effects of some of these compounds on estrogen receptors (ER), the translocation of the estrogen-ER complexes formed in presence of competing substances into the nucleus, as well as the binding of these complexes to DNA. The results of uterotrophic effects of analysed derivatives are in agreement with the influence of these compounds on activity and binding parameters of estrogen receptors. Namely, compounds that show relatively high antiestrogenic activity predominantly increase K-d and inhibit translocation to nuclei of radioactive complexes formed in their presence. On the other hand, compounds that do not significantly change binding parameters of estrogen receptors do not show antiestrogenic effect in in vivo experiments.

Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.

The cloning of the opioid receptors allows the investigation of receptor domains involved in the peptidic and nonpeptidic ligand interaction and activation of the opioid receptors. Receptor chimera studies and mutational analysis of the primary sequences of rite opioid receptors have provided insights into the structural domains required for the ligand recognition and receptor activation. In the current review, we examine the current reports on the possible involvement of extracellular domains and transmembrane domains in the high-affinity binding of peptidic and nonpeptidic ligands to the opioid receptor. The structural requirement for the receptors' selectivity toward different ligands is discussed. The receptor domains involved in the activation and subsequent cellular regulation of the receptors' activities as determined hy mutational analysis will also be discussed Finally, the validity of the conclusions based on single amino acid mutations is examined. (C) 2000 John Wiley & Sons, Inc.