The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.
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ABSTRACT:
SUBMITTER: Krug U
PROVIDER: S-EPMC7736470 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.
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Project description:We briefly survey the current knowledge and concepts regarding structure and function of the neuropeptide Y Y2 receptor and its agonists, especially as related to pharmacology of the receptor and its roles in pathological processes. Specific structural features are considered that could be responsible for the known compartmentalization and participation of the receptor in cell and tissue organization. This is further discussed in relation to changes of levels of the Y2 receptor in pathological conditions (especially in epilepsy and drug abuse), to endocytosis and recycling, and to participation in wound healing, retinopathy and angiogenesis. Properties of the receptor and of Y2 agonists are considered and reviewed in connection to the negative regulation of transmitter release, feeding, mood and social behavior. The possible involvement of the Y2 receptor in diabetes, carcinogenesis and bone formation is also reviewed.
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Project description:G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.
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Project description:The current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate.Salivary samples (n?=?116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value<0.01), and was associated with feeding status (chi square p value ?=? 0.013).Developmental maturation of hypothalamic regulation of feeding behavior is an essential component of oral feeding success in the newborn. NPY2R expression in neonatal saliva is predictive of an immature feeding pattern. It is a clinically relevant biomarker that may be monitored in saliva to improve clinical care and reduce significant feeding-associated morbidities that affect the premature neonate.
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Project description:With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.
| S-EPMC2579264 | biostudies-literature
Project description:The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC(50) values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K(i) values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.
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Project description:Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance (13)C and high-resolution (of 0.01 Hz order) (1)H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal NH(3)(+) group in achatin-I; the de-ionization of NH(3)(+) decreases the bound fraction of the peptide from approximately 15% to nearly none. The electrostatic attraction between the N-terminal positive NH(3)(+) group and the negative PO(4)(-) group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the (13)C chemical shifts (delta) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Delta delta of the (13)C resonance for almost all the carbon sites in achatin-I. The changes Delta delta for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic CO(2)(-) groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic d-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.
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Project description:G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.
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Project description:Adrenergic activation of electrogenic K+ secretion in isolated mucosa from guinea pig distal colon was desensitized by peptide-YY (PYY). Addition of PYY or neuropeptide-Y (NPY) to the bathing solution of mucosae in Ussing chambers suppressed the short-circuit current (Isc) corresponding to electrogenic Cl- secretion, whether stimulated by epinephrine (epi), prostaglandin-E2 (PGE2), or carbachol (CCh). Neither peptide markedly inhibited the large transient component of synergistic secretion (PGE2 + CCh). Sustained Cl- secretory Isc was inhibited approximately 65% by PYY or NPY, with IC50s of 4.1 +/- 0.9 nM and 9.4 +/- 3.8 nM, respectively. This inhibition was eliminated by BIIE0246, an antagonist of the Y2-neuropeptide receptor (Y2-NpR), but not by Y1-NpR antagonist BVD10. Adrenergic sensitivity for activation of K+ secretion in the presence of Y2-NpR blockade by BIIE0246 was (EC50s) 2.9 +/- 1.2 nM for epi and 13.3 +/- 1.0 nM for norepinephrine, approximately fourfold greater than in the presence of PYY. Expression of mRNA for both Y1-NpR and Y2-NpR was indicated by RT-PCR of RNA from colonic mucosa, and protein expression was indicated by immunoblot. Immunoreactivity (ir) for Y1-NpR and Y2-NpR was distinct in basolateral membranes of columnar epithelial cells in the crypts of Lieberkühn as well as intercrypt surface epithelium. Adrenergic nerves in proximity with crypts were detected by ir for dopamine-beta-hydroxylase, and a portion of these nerves also contained NPY(ir). BIIE0246 addition increased secretagog-activated Isc, consistent with in vitro release of either PYY or NPY. Thus PYY and NPY were able to suppress Cl- secretory capacity and desensitize the adrenergic K+ secretory response, providing a direct inhibitory counterbalance against secretory activation.
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Project description:Gene therapy to treat pharmacoresistant temporal lobe epilepsy in humans is now being developed using an AAV vector (CG01) that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2. With this in mind, the present study aimed to provide important preclinical data on the effects of CG01 on the duration of transgene expression, cellular tropism, and potential side effects on body weight and cognitive function. The CG01 vector was administered unilaterally into the dorsal and ventral hippocampus of adult male rats and expression of both transgenes was found to remain elevated without a sign of decline at 6 months post-injection. CG01 appeared to mediate expression selectively in hippocampal neurons, without expression in astrocytes or oligodendrocytes. No effects were seen on body weight as well as on short- or long-term memory as revealed by testing in the Y-maze or Morris water maze tests. Thus these data show that unilateral CG01 vector treatment as future gene therapy in pharmacoresistant temporal lobe epilepsy patients should result in stable and long-term expression predominantly in neurons and be well tolerated without side effects on body weight and cognitive function.
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