Solubilization and characterization of active neuropeptide Y receptors from rabbit kidney

Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.     

Y2 receptor proteins for peptide YY and neuropeptide Y. Characterization as N-linked complex glycoproteins.

Affinity labeling using [125I-Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both M(r) = 50,000 to 60,000 and M(r) = 96,000 to 115,000 [1,2]. In this work the glycoprotein nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N-glycosidase F (PNGase F), and O-glycanase treatment. Only N-glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16-17 kDa units, that is M(r) = 96,000 to 79,000 and M(r) = 60,000 to 44,000, indicating removal of N-linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N-linked complex (sialo)glycoproteins with a minimal core protein size of M(r) = 44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn-linked carbohydrate may be of the tri- or tetra-antennary complex type containing terminal sialic acid residues.     

Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling

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Isolation and sequence of rat peptide YY and neuropeptide Y

Rat peptide YY and rat neuropeptide Y have been isolated in parallel from colon and brain extracts respectively, using salt fractionation, gel filtration chromatography, cation-exchange HPLC, and reverse phase phenyl-silica HPLC. Immunoreactivity was identified using a combination of 3 NPY immunoassays which exhibit differing cross-reactivities for PYY (90%, less than 0.01% and 30% respectively). The yield at the final purification step was 1.2 nmol rPYY and 0.5 nmol rNPY. Half of each purified peptide was subjected to complete microsequence analysis. This showed that while rat NPY was structurally identical to human NPY, the sequence of PYY from rat colon was the same as porcine PYY isolated from extracts of duodenum.     

Solubilization and purification of Na, K-ATPase from the outer medulla of rabbit kidney

There is great interest in the histologic localization of the Na, K-pump in various tissues: histochemical methods in this case work poorly, so a specific antibody against a purified soluble Na, K-ATPase is necessary. We approached this problem with a two-step ionic detergent treatment; the separation of the solubilized enzyme was attempted by gel filtration and ion-exchange chromatography. The gel filtration purified form was active and nearly pure, while the ion-exchange on was purer but inactive. PAGE analyses of the various enzyme forms are presented.     

Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex

This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.     

Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared [125I]PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of [125I]PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of [125I]PYY binding sites throughout the rat brain was seen to be similar to the distribution of [125I]NPY binding sites.     

Aminopyrazoles with high affinity for the human neuropeptide Y5 receptor

1,3-Disubstituted-5-aminopyrazoles were prepared based on a lead compound found through high-throughput screening of our corporate compound library in an assay measuring affinity for the human neuropeptide Y5 receptor. The target compounds were prepared by cyclization of alpha-cyanoketones with appropriate hydrazines, followed by reduction and coupling to various sulfonamido-carboxylic acids. Several of these arylpyrazoles (e.g., 19 and 45) displayed high affinity for the human NPY Y5 receptor (<20nM IC(50)s).     

Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion

Neuropeptide Y (NPY) injected into the paraventricular nucleus (PVN) is known to elicit a powerful feeding response in satiated, brain-cannulated rats [41, 42, 43]. The present experiment investigates the effect of peptide YY (PYY), a structurally-related peptide, on feeding behavior and, in addition, the effects of both PYY and NPY on the pattern of macronutrient selection. Injection of PYY directly into the PVN, in doses ranging from 7.8 to 235 pmol/0.3 μl, caused a strong, dose-dependent stimulation of feeding behavior, as well as a small stimulation of drinking behavior, in satiated rats. The mean latency to eat was 9.3 min, with substantial feeding occurring within 30 min of the injection. At low doses, the increase in feeding was seen predominantly during the first hr. At the highest dose, in contrast, food intake continued to increase progressively over the next few hr, such that by 4 hr postinjection food intake was more than 20 g over vehicle baseline. In 1 hr tests with 3 pure macronutrient (protein, fat and carbohydrate) diets simulataneously available, PYY and NPY (78 pmol/0.3 μl) both elicited a strong and selective increase in carbohydrate consumption, with little or no effect on protein or fat consumption. These results suggest that hypothalamic receptors sensitive to PYY and NPY may participate in the control of carbohydrate consumption.     

Identification of cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY.

Neuropeptide Y (NPY) and peptide YY (PYY) are homologous 36 amino acid amidated peptides that often, but not always, exert similar actions and binding profiles. The present study of cultured cells confirms that both peptides as well as radioiodinated analogs, i.e. 125I-Bolton-Hunter-NPY (125I-BH-NPY) and 125I-peptide YY (125I-PYY), show high affinity to binding sites/receptors of the previously proposed Y1- and Y2-subtypes, selectively expressed by the human neuroblastoma cell lines, SK-N-MC and SK-N-BE(2), respectively. In contrast, bovine adrenal chromaffin cells did not bind 125I-PYY, while displaying high affinity 125I-BH-NPY sites, and may therefore represent a cell type expressing a recently proposed Y3-type of (NPY-preferring) receptors. Several non-labeled fragments/analogs have been used in displacement experiments to further characterize the structural requirements for Y1-, Y2-, and Y3-type binding. In every instance, specific binding was reduced by addition of 5'-guanylylimidodiphosphate [Gpp(NH)p], indicating that the three receptor subtypes belong to the G-protein-coupled superfamily of receptors. Moreover, in both neuroblastoma cell lines, the peptides elicited, with appropriate orders of potency, reduction of forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Finally, NPY-evoked 45Ca2+ influx was observed in SK-N-MC and in chromaffin cells. A common dual coupling mechanism of NPY/PYY receptors, i.e. to reduction of cAMP and to Ca2+ elevation, is therefore suggested to exist, although both phenomena could not be demonstrated in every cell type.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

Peptide YY/neuropeptide Y Y1 receptor expression in the epithelium and mucosal nerves of the human colon.

OBJECTIVES: Peptide YY is an abundant distal gut hormone which regulates secretion, motility, and possibly epithelial proliferation in the gut. Though messenger RNA for the peptide YY Y1 receptor subtype occurs in the basal colonic crypts of humans, peptide YY receptors themselves have not been clearly localized within the adult human gastrointestinal tract. Using an antiserum directed against the C-terminus of the Y1 receptor we determined the actual extent of Y1 receptor protein expression in the human colon in order to identify areas targeted for peptide YY effects and suggest additional physiological roles for PYY in the human gut. RESULTS: Y1 receptor protein expression was seen throughout the colonic epithelium along its basolateral aspect. There was an unexpected dense distribution of Y1 receptor immunoreactivity in varicose fibers within the mucosa. Staining was also noted in nerve fibers of the muscularis mucosae, in the submucous and myenteric plexuses, and in nerves in the muscularis propria. CONCLUSIONS: Widespread distribution of Y1 receptors in the colonic epithelium and mucosal nerve fibers suggests diverse regulatory roles for peptide YY in modulating epithelial function as well as secretomotor reflexes in response to lumenal peptide YY-release signals.     

Identification of a novel 65-kDa cell surface receptor common to pancreatic polypeptide, neuropeptide Y and peptide YY

By affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, we identified a novel cell surface receptor on intact rat cells, which bound, with similar dissociation constants, pancreatic polypeptide (PP), neuropeptide Y (NPY) and peptide YY (PYY), the members of the PP family. The receptor was detected on pancreatic islet and acinar cells, hepatocytes and epithelial cells of the stomach, duodenum and small intestine. Its molecular weight was estimated to be 65,000, and the cross-linking of [125I] labeled ligands was inhibited by an excess of unlabeled PP, NPY or PYY. The results suggest that the 65-kDa molecule is a common receptor for PP family peptides.