©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Biochemical Characterization of Neuropeptide Y Receptor (*)

(Received for publication, November 28, 1994; and in revised form, February 17, 1995)

Sunil J. Wimalawansa (§)

From the Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Neuropeptide Y (NPY) receptors consist of three subtypes, designated NPY(1), NPY(2), and NPY(3). The Y(1) receptor has been cloned. The present study reports the purification of the NPY-Y(2) 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-Y(2) receptor was identified as a monomeric glycoprotein with a molecular mass of 60 kDa. Following deglycosylation, the molecular mass of the Y(2) receptor was decreased to 45 kDa. Although the I-NPY binding to the purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect. Therefore, the purified NPY-Y(2) receptor is probably not associated with G-proteins, but may have intramolecular-free sulfhydryl groups. The specific activity of the isolated NPY-Y(2) receptor is 15.8 nmol/mg of protein. The isolated receptor retained its capacity to bind to I-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 Y(2) subtype as demonstrated by its affinity to C-terminal fragments of NPY, including NPY-(13-36).


INTRODUCTION

Neuropeptide Y (NPY) (^1)is a 36-amino-acid peptide (1, 2) and shares amino acid sequence homology with pancreatic polypeptides (PP) (50% homology) and peptide YY (PYY) (69% homology)(1, 3) . It is a member of the superfamily of peptides consisting of NPY, PP, and PYY(1, 2, 3) . Human NPY and porcine NPY are structurally identical except for the amino acid residue 17 (methionine in human-NPY and leucine in porcine-NPY)(3) . NPY is abundant in the central nervous system of all mammalian species(3, 4, 5) , including human(6) . Although the physiological functions of this peptide are incompletely understood, many pharmacological and behavioral effects of centrally administered NPY and homologous peptides have been reported(3) . These effects include increases in fluid and food intake(7, 8, 9) , changes in sexual behavior (9) and body temperature, decreased motor activity and induced catalepsy, and increased muscle tone(10) . NPY is a potent vasoconstrictor peptide and is present in the heart around the coronary arteries and in the sinoatrial and atrioventricular nodes(11, 12, 13) . NPY is often co-localized and co-released with noradrenaline (14) and modulates catecholaminergic responses in various tissues(11, 13, 15) . As with the other members of the NPY/PP family, NPY alters circadian rhythms when injected into the suprachiasmatic nucleus (16) and elicits numerous physiological responses by specific pre- and postsynaptic receptors.

NPY receptors have been identified throughout the brain. Two classes of NPY receptors with high and low affinity have been found in the porcine brain(17, 18) , but not in the rat brain(19, 20, 21, 22) . Functional and binding studies have indicated three distinct NPY/PYY receptor subtypes, Y(1), Y(2), and Y(3)(15, 18, 23, 24, 25, 26, 27, 28, 29) . Pharmacological characterization of NPY-Y(1) and -Y(2) receptor subtypes is based on the affinity of C-terminal NPY fragments (e.g. NPY-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) ) to the Y(2) receptor, whereas Y(1) receptors interact only with the intact NPY/PYY-(1-36)(26) . Quantitatively, Y(2) receptors are the most abundant NPY receptor subtype in the brain(18, 29, 30, 31) . NPY and its receptors are highly concentrated in the limbic and olfactory systems, hypothalamo-neuro-hypophyseal tract, corpus striatum, and in cerebral cortex(17, 18, 32, 33) . Based on electrophysiological and competitive binding data, NPY-Y(2) receptors have been found in the olfactory bulb, superficial layers of the cortex, and hippocampus, localized to prejunctional sites of neurons(31) , and Y(1) receptor in the lateral septum, hypothalamus, thalamic nuclei, and area postrema(4, 6, 19, 32) . In the vasculature, Y(2) receptors are found at a prejunctional site on sympathetic nerves(15) , whereas the postjunctional NPY receptors are either the Y(1) or Y(2) subtype(26, 27, 28, 29, 34) .

NPY and PYY seem to have similar receptors in the brain and other tissues (35) and have been shown to be highly conserved throughout evolution(20) . NPY receptor subtype Y(1), but not Y(2), was cloned and sequenced(23, 36) . Because of the involvement of the Y(2) receptors in neurophysiology (e.g. fluid and food intake, sexual behavior, control of body temperature) and the cardiovascular system, we decided to extend the studies with this particular receptor subtype to further understand its involvement in pathophysiology and to aid in possible future drug development. Solubilization and analytical scale purification of NPY receptors have been reported previously(18, 37) . The present study was undertaken to isolate and purify the NPY-Y(2) receptor with a view to raising monoclonal antibodies and to obtain partial amino acid sequences for cloning the Y(2) receptor gene to understand the actions of NPY at the molecular level. Pig brain was chosen as the starting material because it is rich in NPY-Y(2) receptor and we could obtain unlimited supplies of fresh material. This paper reports the first quantitative isolation, purification, and biochemical characterization of NPY-Y(2) receptors from porcine brain.


EXPERIMENTAL PROCEDURES

Materials

NPY-(1-36) and its fragments were synthesized using t-butoxycarbonyl chemistry in solid-phase technology on an ABI-430A automated peptide synthesizer (Applied Biosystems). Synthetic NPY was purified by ion exchange chromatography, followed by reversed phase-HPLC and fully characterized by fast atom bombardment mass spectrometry, amino acid sequencing, and amino acid analysis. Monoiodinated I-Tyr-NPY was prepared by a modified chloramine T method and purified by reversed phase-HPLC. Bolton-Hunter-labeled [I-Lys^4]NPY was purchased from Amersham International (specific activity 74 TBq/mmol (2000 Ci/mmol)). Affi-Gel 10 and polyacrylamide gel electrophoresis reagents were from Bio-Rad, disuccinimidyl suberate from Pierce, molecular weight markers and protease inhibitors from Sigma, and wheat germ agglutinin-agarose (WGA) and concanavalin A (ConA) from Boehringer Mannheim. All other chemicals and reagents were of reagent grade and were from Sigma. For the final purification procedure, 1000 pig brains weighing 250 kg were collected in batches from a slaughterhouse.

Preparation of Neural Membranes

Pig brains were removed from decapitated heads, immediately placed in dry ice, and stored at -70 °C. Experiments were carried out by thawing six pig brains at a time. Spinal cord, meninges, cerebellum, and some white matter were removed because these tissues are not rich with NPY-Y(2) receptors. Brain tissues were dissected on ice and quickly homogenized after addition of 5 volumes of freshly prepared ice-cold homogenization buffer containing 50 mM HEPES, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 320 mM sucrose (pH 7.4). The dissection time and temperature were kept to a minimum to prevent receptor losses due to enzyme activation. Homogenized membranes were centrifuged at 1000 g for 10 min at 4 °C, and the supernatants were collected. The pellet was then rehomogenized in 3 volumes of fresh homogenization buffer, and the supernatants were pooled and centrifuged at 30,000 g for 45 min at 4 °C. The membrane-rich pellets were kept for solubilization and assay for receptor activity. Identical results were obtained on I-NPY receptor binding activity with the 100,000 g microsomal pellet and with the cerebral membranes prepared on a discontinuous sucrose gradient to give a synaptosomal membrane preparation compared with 30,000 g crude membrane pellet. Therefore, for convenience and for the handling of a large quantity of material, 30,000 g crude membranes were used for the NPY receptor purification.

Solubilization of Membrane-bound Receptors

Optimum conditions for solubilization of active NPY receptors were assessed by treating cerebral membranes with different detergents of various concentrations. Cerebral membranes were resuspended at 2 mg/ml in 50 mM HEPES, 1 mM MgCl(2) in the presence of 1 mM PMSF, and aprotinin, 100 units/ml. After addition of detergent, the mixtures were gently rocked for 60 min at 4 °C. The resultant mixture was then centrifuged at 30,000 g for 90 min at 4 °C in an MSE cooling centrifuge. The supernatant was removed and assayed for I-NPY binding and for protein content. Several widely used detergents, including Triton X-100, digitonin, sodium cholate, Zwittergent 3-14, Lubrol PX, CHAPS, and n-octyl-beta-D-glucoside, were examined for efficiency of solubilization of NPY receptors. The ability of NPY receptors to bind to I-NPY was measured under various storage conditions with protease inhibitors, such as PMSF, bestatin, and aprotinin, and other protectants, such as glycerol. The stability of solubilized NPY receptors were assessed because the solubilized receptors are more prone to degradation and denaturation than are membrane-bound receptors.

I-NPY Binding Assay for Membrane-bound and Solubilized Receptors

NPY was iodinated with a modified chloramine T method, purified by reversed phase-HPLC, and assayed as described previously(18) . NPY receptor activity in the cerebral membrane was measured by a binding assay with I-NPY. In brief, 100-µl membrane suspensions (2.0 mg of protein/ml) were incubated with 30 pMI-NPY in a 50 mM HEPES buffer (pH 7.0), containing 5 mM KCl, 1 mM PMSF, 200 kallikrein inactivating units of aprotinin/ml in the presence of 0.2% (w/v) heat-inactivated bovine serum albumin, in triplicate for 120 min at 4 °C, while they rocked in a total volume of 200 µl. The label-bound membranes were separated by centrifugation at 11,000 g for 2 min at 4 °C in a microcentrifuge.

I-NPY-bound solubilized receptors were separated by adding 125 µl of 0.5% human -globulin and then 500 µl of 30% polyethylene glycol. The assay tubes were vortexed and incubated for an additional 10-min period at 4 °C; 1 ml of 15% polyethylene glycol was then added, vortexed, and centrifuged at 5000 g for 20 min at 4 °C. The tubes were decanted, and the pellet was counted for the bound I-NPY activity. Identical incubations were carried out omitting the detergent extracts to obtain background counts, and these were subtracted from the total and the nonspecific binding before analysis. The difference in count in the absence and presence of excess unlabeled NPY (0.5 µM) was taken as the specific binding activity.

Degradation of I-NPY was assessed by precipitation with trichloroacetic acid and was routinely <5% at the end of the incubation. Competitive binding data were analyzed by computerized Scatchard analysis(38) . Specificity of I-NPY binding to membranes and solubilized receptors was examined by competition of NPY, NPY fragments, and various other peptides in the presence of I-NPY. In the experiments in which competitive binding was assessed, concentration of unlabeled NPY and related peptides varied from 0 to 1 µM. Membrane- and octyl glucoside-solubilized NPY receptors were incubated with increasing concentrations of I-NPY in assay buffer, in triplicate for 120 min at 4 °C to assess the kinetics of I-NPY receptor binding.

Protein Concentration

The influence of protein concentration and the detergent:protein ratio in the efficiency of solubilization of NPY receptors were examined to determine the optimum membrane protein:detergent concentration. Protein was estimated using a dye-binding method (39) and a microplate reader to facilitate measurement of a large number of samples of small volume (e.g. 50 µl).

Hydrophobic Interaction (HIC) and Ion Exchange (IEC) Chromatography

The solubilized NPY receptor solution was loaded onto a phenyl-Sepharose CL-4B HIC column. The bound receptors were eluted with an inverse salt gradient, and the fractions containing NPY receptors were pooled and applied to an ion exchange column (IEC). Preliminary experiments suggested that NPY receptors with a net positive charge can be separated on cation exchange columns below their isoelectric point. We decided to use the HIC and IEC columns because of the capacity of these columns to handle a large quantity of solubilized material, although the recovery of NPY receptors was not optimal. The chromatographic fractions (11 to 18) from HIC containing NPY receptor activity were pooled and dialyzed against HEPES buffer (10 mM, pH 7.5). The dialyzed material from 2 to 3 HICs was pooled and applied to a QAE-Sephadex A-25 ion exchange column. Bound NPY receptors were eluted with an increasing ionic strength of NaCl up to 1 M. The fractions (11 to 16) containing specific NPY binding activity were pooled and dialyzed before being used in specific NPY-affinity chromatography.

Specific Affinity Chromatography of NPY Receptor

Affinity columns were prepared by covalently coupling synthetic NPY-(1-36) to activated agarose (Affi-Gel 10) (1 mg of NPY/ml of Affi-Gel) because this was suitable for peptides having their isoelectric points at neutral to basic pH, as in the case of NPY. The optimum pH facilitating a higher coupling efficiency of NPY to Affi-Gel was investigated by carrying out the coupling reaction at 4 °C for 4 h and using 50 mM acetate buffer of pH values from 5 to 8. A known amount of I-NPY was also added to the unlabeled NPY during coupling to Affi-Gel, and the percentage of incorporation was determined by counting the radioactivity of the NPY-Affi-Gel complex after washing the matrix three times. Pooled, dialyzed fractions containing NPY receptors from 3 to 4 IECs were loaded onto the specific NPY-affinity column. Columns were washed thoroughly with 10 mM HEPES buffer (pH 7.4), followed by glass-distilled water, and then eluted with 1 mM acetic acid (pH 3.0). Eluted material was neutralized immediately with 1 M NH(4)HCO(3), and an aliquot was taken out to estimate I-NPY binding activity. The remainder was then reloaded onto a second specific NPY-affinity column and eluted as described above. Fold of purification at each step was calculated by the improvement obtained in the quantity of the NPY receptor to protein ratio.

Chemical Cross-linking of I-NPY to Cerebral NPY Receptors

Crude cerebral membranes (30,000 g pellet) were incubated at 1.0 mg of protein/ml in 50 mM HEPES buffer (pH 7.4), containing 5 mmol of KCl, 1 mM MgCl(2), and 1 mM PMSF with 50 pMI-NPY, for 120 min at 4 °C in the presence or absence of 50 nM unlabeled NPY. Cross-linking experiments were also performed in the presence of 0.5 µM calcitonin, katacalcin, amylin, and calcitonin gene-related peptide (CGRP), 50 nM NPY-(1-36), PYY, PP, and NPY-(13-36). The receptor-bound radioligand was separated by centrifugation, washed, and resuspended in 1.0 ml of the same buffer. Disuccinimidyl suberate was freshly dissolved in dimethyl sulfoxide, added to give a final concentration of 1 mM, and was cross-linked for 30 min at 4 °C. The reaction was then quenched with 1.0 ml of ice-cold Tris/HCl buffer (pH 7.4). The cross-linked receptor-ligand complex was pelleted by centrifugation, washed, and solubilized with 1% octyl glucoside, 50 µl of 10% sodium dodecyl sulfate (SDS), and 20 µl of 20% glycerol in the presence or absence of 2-mercaptoethanol. Similar experiments were conducted with the affinity-purified receptor with the exception of inclusion of additional C-terminal NPY fragments NPY-(16-36), NPY-(22-36), and PYY-(1-36). Cross-linked I-NPY-receptor complexes were concentrated using Ultrafree C3 (Millipore) and subjected to electrophoresis on slab gels containing 10% polyacrylamide (SDS-PAGE) at a constant voltage of 140 V for 2 h(40) . The gels were stained with Coomassie Brilliant Blue, destained, and dried. Autoradiographs were obtained by exposing the dried gel to Kodak X-Omat AR film with enhancing screens at -70 °C.

Lectin Affinity Chromatography

Fresh cerebral membranes and I-NPY cross-linked membranes were solubilized with octyl glucoside. Solubilized receptors were examined for adsorption on immobilized lectins. Samples were loaded onto WGA or ConA lectin columns pre-equilibrated with binding buffer. Columns were washed, and the adsorbed lectin-bound material was eluted by adding the appropriate competitive monosaccharide-alpha-methyl-D-glucoside on ConA columns and N-acetyl-D-glucosamine (1 M) on WGA columns, respectively. In the case of receptors cross-linked to I-NPY, the radioactivity was counted on the eluted material after precipitation by polyethylene glycol, whereas with a solubilized uncross-linked receptor, binding activity was assessed with I-NPY.

Lectin chromatography was not used during purification of NPY receptor because of its poor adsorption, as well as the inadequate recoveries from the lectin affinity columns. For assessment of the quantity of the glycosylation component, solubilized cross-linked receptors and the purified receptors were deglycosylated (37 °C for 3 h) with protease-free endoglycosidase F (0.5 milliunit) and O-glyconase (2 milliunits) enzymes (Glycoanalysis, MA). Control samples were treated in an identical fashion, but without enzymes. These samples were then concentrated by chloroform-methanol precipitation (41) and subjected to SDS-PAGE as described above. The cross-linked NPY receptor was visualized with autoradiography and purified NPY-Y(2) receptor by Western blotting, with NPY-Y(2)-specific anti-receptor monoclonal antibodies.

HPLC-Gel Permeation Chromatography

HPLC-gel permeation chromatography was used to obtain an index of the molecular size/configuration of the NPY receptor. Highly purified NPY receptors were loaded onto a SW3000G column and eluted with pH 5.5, 0.2 M NH(4)HCO(3) buffer containing 0.2% octyl glucoside at room temperature. The chromatographic fractions were then assayed for I-NPY binding activity. Solubilized cross-linked NPY-receptor complexes (in the absence of NPY and in the presence of NPY, PYY, NPY-(13-36), or CGRP) were also studied in a similar fashion in separate chromatographs, and radioactivity in the fractions was counted. Molecular weight markers were chromatographed separately on the same column, and the relative elution positions of the receptor were calculated (K)(42) .


RESULTS

The Effect of Different Detergents on the Solubilization of NPY Binding Activity

The criterion of optimal solubilization was judged as the one that yielded the highest I-NPY specific binding activity while releasing the least amount of total protein (i.e.I-NPY binding/mg of protein). The specific binding of solubilized NPY receptors and the percentage of the total protein solubilized with each detergent were 0.5% and 45%, 2.6% and 49%, 4.2% and 40%, and 8.5% and 38% for Triton, digitonin, CHAPS, and octyl glucoside, respectively. Solubilization with octyl glucoside was superior to that obtained with Triton, CHAPS, and cholate. Among the various concentrations of octyl glucoside examined, a 1% final concentration was selected because it solubilized the maximum quantity of intact NPY receptors and the least proteins. In a similar fashion, we examined the effects of various concentrations of K(2)HPO(4) and the octyl glucoside:K(2)HPO(4) ratio on NPY receptor solubilization and found that optimum solubilization of NPY receptors was achieved with 1% octyl glucoside and 125 mM K(2)HPO(4) (Fig. 1). Concentrations below this dramatically decreased the solubilization efficiency. The maximum specific activity was detected at the detergent:protein ratio of 6:1 (mg/mg).


Figure 1: Effects of detergent concentrations on the solubilization of NPY receptors and membrane protein by 1% octyl glucoside. Membranes were diluted with buffers containing various concentrations of phosphates and centrifuged at 30,000 g for 90 min at 4 °C. The resulting supernatant and pellet were tested for their ability to bind to I-NPY and compared with that of the membrane-bound receptors (n = 6). Concentrations of K(2)HPO(4) used were: 1000 mM, 500 mM, 250 mM, 125 mM, and 62.5 mM, whereas octyl glucoside concentration remained at 1%. The yield of solubilized protein (shaded bars) and NPY receptors (hatched bars) is expressed as a percentage of the amount of proteins or receptors in the fresh membrane preparation, as determined by protein assay and I-NPY binding, respectively, as described under ``Experimental Procedures.''



Stability of NPY Receptor

Pilot experiments carried out after optimization of the solubilization procedure indicated that approximately 200 separate solubilization experiments (6 brains/experiment) were required to acquire an adequate quantity of NPY receptor for obtaining partial amino acid sequences of the NPY-Y(2) receptor. Therefore, it was important to maintain the stability and the integrity of the solubilized NPY-Y(2) receptor during storage as well as throughout the purification procedure (i.e. to be able to utilize a specific NPY-affinity purification step). When the stability of NPY receptors was tested at -20 °C for a period of 1 month with different detergent concentrations, it showed that the receptors were most stable in 1% octyl glucoside, 125 mM K(2)HPO(4) buffer than in higher concentrations of the detergent. Loss of activity was proportional to the increase in concentration of the detergent. However, a combination of protease inhibitors (PMSF, bestatin, and aprotinin) and 20% glycerol preserved I-NPY binding activity and permitted storage of the samples for 2 to 3 days at 4 °C or up to 35 days at -70 °C. This finding was crucial because HIC and IEC steps each took several hours (usually overnight) to complete. The presence of 1 mM dithiothreitol increased the stability of NPY receptors during freezing and thawing. Irrespective of temperature of storage, the activity of the solubilized NPY receptor tends to decrease with time and also is affected by temperature and ionic concentration of the buffer.

Receptor Binding

I-NPY binding to both crude membranes and the solubilized receptors was specific and time- and temperature-dependent. At 4 °C, the binding reached a steady state after 120 min and was maintained up to 3 h. Binding properties of octyl glucoside-solubilized receptors were determined and compared with those of membrane-bound receptors with competitive binding of I-NPY. Scatchard analysis of the data obtained after increasing the concentration of I-NPY suggested that the binding of NPY to its receptors was saturable and disclosed a curvilinear plot with upward concavity that suggested receptor heterogeneity as previously reported(18, 21) . The specificity of solubilized receptors was unchanged, and the binding data for solubilized receptors were similar to the curvilinear plots obtained for membrane-bound receptors(18) . Although the detergent itself may interact with the ligand-receptor complex and affect the Scatchard data, the dissociation constants (K) obtained from the membrane-bound and solubilized NPY receptors are comparable (Table 1)(18) . However, the maximum binding capacity (B(max)) expressed as femtomoles/mg of protein is doubled in the solubilized fraction, but retained the same pharmacological specificity as those of membrane-bound receptors ( Table 1and II).



NPY and PYY displaced bound I-NPY from the solubilized receptors with half-maximal concentrations (ED) of 1.1 10 and 1.6 10, respectively. Structurally related peptides porcine-, avian-, and bovine-PP inhibited I-NPY receptor binding only over 800, 2,500, and 15,000 molar excess, respectively. A number of other peptides, such as vasoactive intestinal peptide, cholecystokinin-8, insulin, growth hormone, calcitonin, amylin, and CGRP were totally ineffective. The C-terminal fragments of NPY (e.g. NPY-(13-36)) displaced bound I-NPY from membrane-bound, solubilized, and purified NPY receptors in a dose-dependent manner (Table 2). However, the N-terminal fragment(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) and the mid-molecule(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) of NPY were ineffective.



Specificity of NPY Receptor

The radioactivity remaining on the membrane-bound and solubilized cross-linked ligand-bound receptor is shown in Fig. 2. The total binding was found to be 30% of the total counts, which may represent both cross-linked and specifically bound I-NPY. The nonspecific binding was <5% in both solubilized and membrane-bound receptors. The addition of 0.5 µM (10^4-fold excess) salmon calcitonin, CGRP, katacalcin, amylin, porcine-PP, and a number of other peptides to the solubilized receptor had no effect on I-NPY binding (Fig. 2).


Figure 2: The specificity of NPY receptors: membrane-bound (hatched bars) and solubilized receptors (shaded bars) incubated with I-NPY and cross-linked using disuccinimidyl suberate. The free unreacted tracer was separated by centrifugation or precipitation of the I-NPY-receptor complex by polyethylene glycol, and the radioactivity of the pellet (total binding) was counted. In determining the nonspecific binding and cross-reactivity, various unlabeled ligands such as NPY, porcine pancreatic polypeptide (PP), katacalcin (KC), calcitonin (CT), CGRP, and insulin were incubated with I-NPY, and the radioactivity of the resulting pellets was determined (n = 5 for each column). Total binding of I-NPY after cross-linking to fresh membrane receptors (hatched bars) and solubilized receptors (shaded bars). The figure shows that displacement of I-NPY was determined in the presence of 50 nM NPY (i.e. nonspecific binding) and of 0.5 µM katacalcin (KC), calcitonin (CT), and calcitonin gene-related peptide (CGRP).



I-NPY binding to the membrane-bound and solubilized NPY receptors was inhibited in a dose-dependent fashion by guanine nucleotides Gpp(NH)p, GTP, and GDP, but not by GMP, as has previously been reported(21, 34, 43) . I-NPY receptor binding was decreased by 80% in the presence of N-ethylmaleimide. With the exception of GMP, these compounds increased the rate of dissociation of the receptor-bound I-NPY and decreased their affinity for the high affinity sites. Similar effects have been reported previously for NPY receptor preparations from porcine brain (44) and rabbit kidney(45) . These data suggest that the solubilized NPY-receptor complex includes a guanine nucleotide-binding protein (G-protein), but the guanine nucleotide effect was lost after the IEC step during purification. The lack of effect of these compounds on the I-NPY binding to the final isolated and purified NPY receptors suggests that either G-protein may not be a part of the purified receptor or that structural change of the protein had occurred. Furthermore, C-terminal fragments of NPY competed with the purified NPY receptor and indicated that the identity was the Y(2) receptor.

Isolation and Characterization of the NPY Receptor

The solubilized receptor preparation was purified sequentially by HIC and IEC. I-NPY binding activity was eluted as a single, but broad, band of several chromatographic fractions in both methods (Fig. 3). That this band coincided with a large quantity of protein suggested that receptors were far from pure. Nevertheless, sequential use of HIC and IEC allowed elimination of the majority of lipids and a large proportion of unwanted proteins, thus permitting the use of the resultant material directly on the specific NPY-affinity columns. Lectin affinity chromatography showed NPY receptors were readily adsorbed by WGA agarose. Of WGA agarose-bound NPY receptors, 30% were eluted by the addition of 0.2 MN-acetylglucosamine. Receptors were also partially adsorbed by ConA, and 39% of the receptors were desorbed with 1 M alpha-methyl glucoside. Deglycosylation of NPY-Y(2) receptor with endoglycosidase F and O-Glyconase enzymes revealed an apparent molecular mass shift of approximately 15 kDa (Fig. 4).


Figure 3: A, the solubilized receptor preparation was loaded onto a hydrophobic interaction chromatographic (HIC) column at a high salt concentration (1% octyl glucoside in 1 M K(2)HPO(4) (pH 7.4)) to maximize binding. The concentration of the mobile phase ionic strength was then gradually decreased with an inverse gradient up to 1% octyl glucoside in 10 mM K(2)HPO(4) to promote elution. Chromatographic fractions were assayed for I-NPY binding activity () and for protein content (bullet). Mean data from 6 experiments are shown. The broken line shows the salt gradient. B, the ion exchange column (IEC) findings: pooled chromatographic fractions from HIC, containing NPY receptor activity, were subjected to overnight dialysis against Tris-HCl buffer (10 mM, pH 7.5) at 4 °C, then loaded onto an IEC (QAE-Sephadex A-25) at low ionic strength and eluted with increasing ionic strengths of NaCl up to 1 M with 1% octyl glucoside. NPY receptor activity () and protein content (bullet) of the chromatographic fractions were determined. Mean data from 6 experiments are shown. The broken line shows the salt gradient.




Figure 4: A, Autoradiography of cross-linked NPY-Y(2) receptor. Lane a, control; lane b, following treatment with endoglycosidase F and O-Glyconase deglycosylating enzymes.



NPY was readily coupled to Affi-Gel 10 in 50 mM sodium acetate buffer (pH 7). The coupling efficiency of NPY to Affi-Gel gradually increased with the pH of the incubation medium: 80% coupling was achieved at pH 8.0, but only 45% at pH 5.0. The highest binding capacity of solubilized NPY receptors and the highest yield of NPY receptors recovered from the NPY affinity columns was found at the first application. This value was taken as 100% to compare the receptor yields obtained during subsequent usage. The efficiency of Affi-Gel columns in purifying NPY receptors was dramatically decreased after repeated use. After being used 10 and 20 times, the efficiency of the column in isolating NPY receptors decreased to 60% and 20%, respectively. Therefore, a newly prepared NPY-specific Affi-Gel column could be used effectively only up to 10 times (e.g. to purify only 60 pig brains).

It was not possible to generate enough purity of NPY receptors from the solubilisate by HIC, IEC, and lectin affinity chromatography, as compared with specific affinity gel chromatography (Table 3). However, the specific NPY-affinity chromatography step was highly efficient in purifying NPY receptors as demonstrated by a considerable increase in specific activity and the finding of a single protein band corresponding to a molecular mass of 60 kDa on the SDS-PAGE and silver staining (Fig. 5). Affinity cross-linking of I-NPY to brain membranes, before and after solubilization, was performed to determine whether the solubilized NPY receptor retained its structural integrity. Fig. 6shows an autoradiogram of SDS-PAGE after cross-linking I-NPY to membrane proteins and subsequent solubilization. The figure shows a specifically labeled protein with an apparent M(r) of 64,000 (Fig. 6, lane 2), which is comparable to the protein band with silver staining (M(r) 60,000) as shown in Fig. 5. The incorporation of I-NPY into this M(r) = 64,000 band was completely inhibited in the presence of 50 nM unlabeled NPY and partially with NPY-(13-36) (Fig. 6, lanes 1 and 3) and suggested that the labeled protein did indeed represent the solubilized NPY receptor.




Figure 5: SDS-PAGE of isolated and purified NPY receptor. Proteins were visualized with silver stain, and molecular weights were estimated against known molecular weight markers. Lane a, affinity-purified NPY receptors after 1 cycle; lane b, affinity-purified NPY receptors after 2 cycles.




Figure 6: Autoradiogram of I-NPY cross-linked to brain NPY receptors. SDS-PAGE was performed under reducing conditions (in the presence of 2-mercaptoethanol) on 10% acrylamide gels. Each lane represents NPY-Y(2) receptors extracted from approximately 4 to 5 pig brains. The gels were stained with Coomassie Brilliant Blue to visualize molecular weight markers, destained, and dried. Autoradiographs were obtained by exposing dried gels to Kodak X-Omat AR films with enhancing screens at -70 °C. Lane 1, cerebral membranes cross-linked in the presence of 50 nM unlabeled NPY-(1-36); lane 2, in the absence of NPY; and lane 3, in the presence of 50 nM NPY-(13-36). a, 97 kDa; b, 66 kDa; c, 58 kDa; and d, 42 kDa.



When the purified NPY receptors were cross-linked with I-NPY, one could demonstrate the specificity further with the competition with various NPY fragments and confirm the identity of the isolated receptor as the Y(2) subtype (Fig. 7). In the presence of 0.5 µM NPY, I binding to membrane-bound, solubilized, and purified receptor was completely inhibited (Fig. 6, lane 1). After two cycles of specific affinity purification, the final yield of NPY receptors was 3.0 nmol, and the purification efficiency was 0.8 µg of NPY receptors/kg of porcine brain resulting in 10^9-fold purification (Table 3). The total recovery for the purification procedure was 1.4%. The specific activity of the purified NPY-Y(2) receptor was 15.8 nmol/mg of protein, being 16.6 nmol/mg as the calculated theoretical values for the pure NPY-Y(2) receptor.


Figure 7: Autoradiogram of I-NPY cross-linked to affinity-purified NPY receptor (1 cycle). Cross-linked purified NPY receptors were chromatographed on SDS-PAGE and autoradiographed as described in Fig. 6. Each lane represents NPY-Y(2) receptors purified from approximately 15 to 20 pig brains. Lane 1, in the presence of NPY-(16-36); lane 2, NPY-(22-36); lane 3, PYY-(1-36); lane 4, NPY-(1-36); lane 5, control; lane 6, NPY-(13-36); lane 7, NPY-(1-36); lane 8, NPY-(1-22).



Molecular Weight Assessment of the Y(2)Receptor by Size-exclusion HPLC

Fig. 8A demonstrates the size-exclusion HPLC profile (SW3000G column) of the affinity-purified Y(2) receptor. The peak of specific binding activity with I-NPY was eluted around M(r) = 66,000. Fig. 8B illustrates the profiles of cross-linked preparations with I-NPY in the presence and absence of NPY-(1-36) or PYY and Fig. 8C, in the presence of CGRP and NPY-(13-36). That the I-NPY binding was completely abolished in the presence of 50 nM NPY/PYY and partly by 50 nM NPY-(13-36), whereas CGRP and other peptides were completely ineffective, suggested the specificity and identity of the purified NPY-Y(2) receptor.


Figure 8: A, I-NPY binding activity following gel permeation chromatography (SW3000G) of purified NPY receptors. Specific binding activities of the chromatographic fractions were detected by I-NPY binding activity (). B, solubilized cross-linked I-NPY-receptor complex was chromatographed, and radioactivity of each chromatographic fraction was counted. Chromatographic profiles in the absence of () and in the presence of (bullet) 50 nM NPY or PYY-(1-36). C, chromatographic profiles of I-NPY-cross-linked NPY receptors, in the presence of 0.5 µM CGRP, or amylin () and 50 nM NPY-(13-36) (bullet). A solution containing molecular weight markers was chromatographed separately on the same column with a UV detector set at 220 nm to assess the elution positions. Molecular weight markers: a, fructose 6-phosphate (84,000); b, bovine serum albumin (66,000); c, chicken albumin (45,000); d, carbonic anhydrase (29,000); e, cytochrome c (12,400); f, aprotinin (6,400); g, KCl.




DISCUSSION

This report demonstrates the first quantitative isolation, purification, and biochemical characterization of NPY-Y(2) receptor. During purification, it was important to retain the capacity of solubilized receptors to bind specifically to the ligand for subsequent use in the specific affinity column because some detergents denatured receptors with detergent-polypeptide chain aggregates and caused a loss of receptor activity. The binding of I-NPY to solubilized NPY receptors was specific and saturable and exhibited high affinity. The kinetics and optimal conditions for binding of I-NPY to solubilized receptors, such as pH, temperature, and ionic strength, were similar to those of membrane receptors(18, 20, 22) . That N-ethylmaleimide significantly affected the I-NPY binding to the solubilized as well as the isolated NPY receptor suggested the existence of free sulfhydryl groups or intramolecular disulfide bridges or both. Indeed, in previous studies using N-ethylmaleimide and benextramine, the presence of sulfhydryl groups in the Y(2) receptor has been demonstrated(46) .

A gradual loss of affinity of C-terminal NPY fragments was observed with the progressive N-terminal truncation of the NPY ligand (Table 2), which is characteristic of the Y(2) receptor(18, 31) . Previous studies have shown that the brain NPY receptor did not distinguish between NPY and PYY(26, 27, 28, 29) . NPY is found in high concentrations in the brain(2, 3, 4, 5, 6, 29) , whereas PYY is located mainly in the gastrointestinal tract(1, 5, 14, 29) . Thus, the Y(2) receptors in the brain (e.g. hippocampus and cerebral cortex) where a high concentration of NPY is found are probably ``NPY receptors''(26, 30, 37, 47) . Y(2) receptors on the renal tubular cells, where no NPY innervation is found, may be PYY receptors, which responds to circulating PYY(43, 45) . Taken together, that purified NPY receptors specifically interact with C-terminal fragments of NPY gives further evidence that the receptor isolated in this study is the Y(2) subtype of the NPY receptor.

Affinity chromatography has been used extensively to purify neurotransmitter receptors(48, 49) . It is especially useful for the purification of detergent-solubilized membrane proteins. The decreasing ability of the NPY-Affi-Gel column to purify NPY receptors after usage could be explained by hydrolysis of the ligand attached to the insoluble support of agarose and/or the poor mechanical and chemical stability that caused ``bleed off'' of ligand or ligand-receptor conjugates. The efficiency of affinity gel was also decreased by lipids building up on the gel matrix and by being clogged by other particular matter, which could be expected after several chromatograms. However, efficiency of the column was not improved by using several methods of cleaning. Although this problem was minimized by prior use of other separation methods such as HIC or IEC, the best performance of the NPY-Affi-Gel columns was achieved only in the first few runs. HIC, IEC, and lectin affinity chromatography (WGA and ConA) by themselves were insufficient to purify the NPY receptor to homogeneity, but, after the specific affinity chromatographic step, NPY receptors appeared as a single band in SDS-PAGE (Fig. 5). The purity of the isolated NPY receptor was further increased, and the receptors were concentrated by a second cycle of specific affinity chromatography. Highly purified NPY receptors retained their ability to bind specifically to I-NPY.

The molecular mass of the NPY receptor obtained after protein purification was 60 kDa, similar to that obtained after chemical cross-linking ( Fig. 4to 7) and by HPLC gel chromatography (Fig. 8). The previously reported molecular mass of the ``NPY-binding protein,'' after cross-linking studies with bifunctional reagents, varied from 37 kDa to 70 kDa. A common receptor for NPY, PYY, and PP has been shown to be 65 kDa(24) , whereas the molecular mass of NPY receptors has been reported between 52-59 kDa and 37-39 kDa(37, 50, 51) . Cross-linked studies have shown that NPY/PYY receptors have two components, with molecular masses of 70 kDa and 50 kDa representing Y(1) and Y(2) receptor subtypes(18, 26, 51) . There seem to be differences in the size of the Y(2) receptor (between 50 and 67 kDa) as demonstrated by cross-linking studies in various mammalian species(18, 20, 37, 51) . However, the molecular mass of another member of this family, avian pancreatic polypeptide receptors, in chicken and pigs was 67 kDa, whereas the avian-PP receptor in dogs was 85 kDa(44) . These data suggest a possible diversity of this family of NPY receptors in the brain and of species differences. Whether this difference is due to major differences in the amino acid sequences of these proteins, or simply due to various degrees of glycosylation (Fig. 4), is yet to be determined.

When I-NPY was covalently attached to membrane proteins, solubilized, and subjected to PAGE or cross-linked to solubilized proteins, the same M(r) = 60,000 protein band was observed as in the native cerebral membranes. This finding indicates that the solubilized NPY receptor retained its structural integrity after solubilization and gives further evidence that I-NPY binds to the functional NPY receptor. Using nondenaturing solvents in HPLC gel permeation chromatography revealed a slightly higher molecualr mass of the purified NPY receptor (about 66 kDa), in contrast to that seen on SDS-PAGE-protein staining (60 kDa), and by I-NPY cross-linking studies with disuccinimidyl suberate (64 kDa) ( Fig. 5to 8). We have used this isolated NPY-Y(2) receptor for raising monoclonal antibodies. Therefore, in the future, isolation of the Y(2) receptor can be achieved with immunoaffinity purification using this specific antibody.

NPY receptors in the brain are similar to APP and PYY receptors, in that they also bear carbohydrate moieties, including mannose. The lectin adsorption studies and change of the M(r) after deglycosylation (Fig. 4) suggested the glycoprotein nature of the brain NPY receptor, as in NPY-Y(1) and many other peptide receptors(49, 52) . Deglycosylation experiments suggested that glycosylated products are responsible for approximately 25% of the M(r) of the intact NPY-Y(2) receptor. That receptor binding was markedly decreased in the presence of the alkylating agent N-ethylmaleimide suggests the importance of free sulfhydryl groups for binding within the Y(2) receptor (46) . A similar effect was previously reported with avian PP receptors (53) . The M(r) remains the same in the presence of reducing agents such as 2-mercaptoethanol and indicates that the receptor consists only of one subunit. These results give added evidence of the nature, identity, and integrity of both the solubilized and purified NPY-Y(2) receptor. In summary, using a preparative scale purification, NPY-Y(2) receptor was quantitatively isolated and then biochemically characterized. The use of receptor autoradiography, protein staining of purified NPY receptors, and gel permeation chromatography of NPY receptors revealed that the NPY-Y(2) receptor is a monomer of the 60-kDa glycoprotein. This isolated 60-kDa protein had a specific activity of 15.8 nmol/mg of protein which approached the theoretical maximum of 16.6 nmol/mg, giving further evidence of the purity of the isolated NPY-Y(2) receptor.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Hypertension and Vascular Research Laboratory, Division of General Internal Medicine, Dept. of Internal Medicine, The University of Texas Medical Branch, 301 University Blvd., Room 8.104, Medical Research Bldg., Galveston, TX 77555-1065. Tel.: 409-747-1860; Fax: 409-747-1861.

^1
The abbreviations used are: NPY, neuropeptide Y; PYY, peptide YY; PP, pancreatic polypeptide; HPLC, high performance liquid chromatography; WGA, wheat germ agglutinin; ConA, concanavalin A; PMSF, phenylmethylsulfonyl fluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; HIC, hydrophobic interaction; IEC, ion exchange chromatography; CGRP, calcitonin gene-related peptide; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

I am grateful to Dr. Richard Bukoski and Dr. Yogesh Awasthi at the University of Texas Medical Branch at Galveston for critical review of this manuscript. The technical assistance of F. Zhang is greatly appreciated.


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