Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

Simple and efficient identification of chromatin modifying complexes and characterization of complex composition.

Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged Wdr82. Purified proteins were analyzed by SDS-PAGE and specific protein bands were identified by mass spectrometry. Subsequently, purified proteins were fractionated on sucrose gradient equilibrium centrifugation to determine overall composition of each identified complex. We describe here simple and efficient approaches for the identification of chromatin modifying complexes and subsequent characterization of complex composition.

Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding.

We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.

Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production.

The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 µg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.

A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment.

Although highly resolved crystal structures of G protein-coupled receptors have become available within the last decade, the need for studying these molecules in their natural membrane environment, where the molecules are rather dynamic, has been widely appreciated. Solid-state NMR spectroscopy is an excellent method to study structure and dynamics of membrane proteins in their native lipid environment. We developed a reconstitution protocol for the uniformly (15)N labeled Y(2) receptor into a bicelle-like lipid structure with high yields suitable for NMR studies. Milligram quantities of target protein were expressed in Escherichia coli using an optimized fermentation process in defined medium yielding in over 10mg/L medium of purified Y(2) receptor solubilized in SDS micelles. The structural integrity of the receptor molecules was strongly increased through refolding and subsequent reconstitution into phospholipid membranes. Specific ligand binding to the integrated receptor was determined using radioligand affinity assay. Further, by NMR measurement a dispersion of the (15)N signals comparable to native rhodopsin was shown. The efficiency of the reconstitution could also be inferred from the fact that reasonable (13)C NMR spectra at natural abundance could be acquired.

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR.

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)(6) tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region.

The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a K(d) of 0.80 +/- 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a K(d) of 0.14 +/- 0.01 nm (mean +/- SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.

Multiplicity of neuropeptide Y receptors: cloning of a third distinct subtype in the zebrafish.

Five different receptor subtypes for neuropeptide Y (NPY) have recently been cloned in mammals. We have discovered three distinct subtypes by PCR in the zebrafish, Danio rerio, and describe here one of these called zYc. The protein sequence identity is 46-51% to mammalian subtypes Y1, Y4 and Y6 and to zebrafish Ya, i.e., the same degree of identity as these subtypes display to one another. The identity to zYb is higher, 75%, indicating that zYb and zYc share a more recent ancestor. The zYc receptor binds NPY and PYY (peptide YY) from mammals as well as zebrafish with high affinities and has a Kd of 16 pM for 125I-pPYY. The pharmacological profile is similar to, but distinct-from, mammalian Y1. zYc inhibits cAMP synthesis. This work suggests that NPY has more receptor subtypes than any other peptide that binds to G protein-coupled receptors. Work is in progress to see if the zebrafish receptors are present in mammals.

Purification and biochemical characterization of neuropeptide Y2 receptor.

Neuropeptide Y (NPY) receptors consist of three subtypes, designated NPY1, NPY2, and NPY3. The Y1 receptor has been cloned. The present study reports the purification of the NPY-Y2 receptor from porcine brain and its biochemical characterization. NPY receptors were solubilized and purified by sequential hydrophobic interaction, ion exchange, and NPY-affinity chromatography. By use of SDS-polyacrylamide gel electrophoresis, high performance liquid chromatography gel permeation chromatography, and chemical cross-linking studies, the affinity-purified brain NPY-Y2 receptor was identified as a monomeric glycoprotein with a molecular mass of 60 kDa. Following deglycosylation, the molecular mass of the Y2 receptor was decreased to 45 kDa. Although the 125I-NPY binding to the purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect. Therefore, the purified NPY-Y2 receptor is probably not associated with G-proteins, but may have intramolecular-free sulfhydryl groups. The specific activity of the isolated NPY-Y2 receptor is 15.8 nmol/mg of protein. The isolated receptor retained its capacity to bind to 125I-NPY, specific to NPY and peptide YY, and showed no cross-reactivity with any other peptides. Highly purified (10(9)-fold purification) NPY receptor from the brain was identified as the Y2 subtype as demonstrated by its affinity to C-terminal fragments of NPY, including NPY-(13-36).

Characterization of the human Y1 neuropeptide Y receptor expressed in insect cells.

We have expressed the human Y1 NPY receptor in insect cells using a recombinant baculovirus (BacY1). Non-linear curve fitting of competition binding data indicates the presence of 500,000-750,000 saturable NPY binding sites per cell. The affinity of the recombinant Y1 receptor for NPY (Kd = 0.38 +/- 0.8 nM) was identical to the natural receptor. We used a foreign epitope to characterize, immunopurify, and localize the recombinant protein. Cross-linking experiments identified a 65 kDa band as the major NPY binding species. Confocal microscopy indicated that although some recombinant proteins are detectable as early as 12 h post-infection, significant expression at the cell surface is only seen 24-48 h post-infection. We also describe a procedure to treat infected Sf21 cells in such a way that they can be frozen and stored at -80 degrees C for many months before being used for binding studies.

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain.

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 x 10(5) cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: 1) electrophysiological responses and 2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20-70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin.