zy zyx zyxw Comparison of the Neuropeptide Y Receptor in the Rat Brain and Intestine"9b IAN L TAYLOR, PETER J MANNON, GREGORY G HEINTZ, LYNN M KAISER, AND TOAN D NGUYEN Duke University Medical Center P.O Box3913 Durham, North Carolina 27710 INTRODUCTION Neuropeptide Y (NPY), together with pancreatic polypeptide (PP) and peptide YY (PYY), constitute a family of structurally related peptides all of which contain 36 amino acids and have a similar tertiary structure.'-4 NPY has neurotransmitter and neuromodCentral adminulator functions in the central, peripheral and enteric nervous istration of NPY increases food intake,s produces hypotension, bradypnea and EEG synchronization.6 and shifts circadian rhythm^.^ NPY applied to intestinal mucosa mounted in Ussing chambers inhibits ion transport with an EC,, of 10-30 nM;*." it inhibits transmural electrical potential and short circuit current, increases mucosal-toserosal Na+ and C1- fluxes, and reduces serosal-to-mucosal C1- fluxes.*-" This inhibitory action is only demonstrable when NPY is added to the serosal (but not luminal) side of the intestinal epithelium These observations correlate with the localization of NPY to intrinsic nerves within the enteric nervous system that end in close proximity to the laterobasal region of the intestinal epithelial cell We have used a recently described method that allowed isolation of intestinal serosal latero-basal membranes (LBM) free from contamination with luminal brush border membranes (BBM), and intracellular endoplasmic reticulum (ER) and Golgi vesiclesi2 to demonstrate that NPY binds preferentially to the serosal LBM of the enterocyte In addition we have cross-linked radiolabelled NPY to its intestinal receptor, and compared the resulting complexes observed on SDS-polyacrylamide gel electrophoresis with the pattern observed after cross-linking the brain receptor zyx zyxwvu zyxwvu T h i s project was supported by funds from the Department of Veterans Affairs Dr Toan Nguyen is the recipient of a FIRST award (DK 40506) and Dr Ian Taylor of a R01 Grant (DK 38216)from the National Institutes of Health bAbbreviations used: NPY: neuropeptide Y; PYY: peptide YY; PP: pancreatic polypeptide; HPLC: high performance liquid chromatography; ER: endoplasmic reticulum; LBM: laterobasal membranes; BBM: brush border membranes; HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; BSA: bovine serum albumin; DlT: dithiothreitol; SDS: sodium dodecyl sulfate; PMSF: phenylmethylsulfonyl fluoride; DSS: disuccinimido suberate; EDTA: ethylenediaminetetraacetic acid; PAGE: polyacrylamide gel electrophoresis; lacto-NPY ,: lactoperoxidase labelled NPY contained in the first radioactive peak isolated on HPLC; IODO-GEN-NPY,: IOW-GEN labelled NPY contained in the second radioactive peak isolated on HPLC; B-H-NPY: Bolton-Hunter labelled NPY 48 zyxwvutsrq zyxwvuts zyxwvu TAYLOR el al.: NPY RECEPTOR zyxw zyxwv zy zyx 49 METHODS Chemicals and Reagents Synthetic NPY, PYY and PP were urchased from Peninsula Laboratories (Belmont, CA) Bolton-Hunter labelled ['2sI-Lys ] NPY (BH-NPY) was purchased from New England Nuclear (specific activity 2200 Cilrnmol) For some experiments one or more of the or IODO-GEN, l and five tyrosines in NPY was radioiodinated using lactoperoxida~e'~ the resulting radiolabelled species purified on high performance chromatogra h (HPLC) l4 Lactoperoxidase was purchased from Calbiochem (Los Angeles, CA), Na7 from Amersham C o p (Arlington Heights, IL), polyacrylamide gel electrophoresis reagents from Bio-Rad (Richmond, CA) and molecular weight standards for gel electrophoresis from Pharmacia (Pistcataway, NJ) Triton-X-100 was from J.T Baker (Phillipsburg, NJ), and all the protease inhibitors (antipain, chymostatin, pepstatin, leupeptin phenylmethylsufonyl fluoride (PMSF), ovomucoid trypsin inhibitor, bacitracin, aprotinin) were from Sigma The crosslinking agent disuccinimido suberate (DSS) was purchased from Pierce (Rockford, IL) X-ray film (XAR-2) was from Eastman Kodak (Rochester, NY) All other chemicals used were of reagent grade B Intestinal Cell Membrane Fraclionatton Membranes were prepared from intestinal epithelial cells dissociated from the jejunum and ileum of fasted male Sprague Dawley rats (-300 gm) using previously described methods.', Protease inhibitors were added during cell dissociation (2.5 kg/d each of antipain, chymostatin, pepstatin, leupeptin, ovomucoid trypsin inhibitor, and mM PMSF) and during the first homogenization (25 kglml each of antipain, chymostatin, pepstatin, leupeptin, ovomucoid trypsin inhibitor, and mM PMSF) Differential centrifugation was used to yield a pellet (P,) that contained mostly ER-Golgi and LBM; precipitation of ER-Golgi with mM CaCI, combined with differential centrifugation yielded a pellet (P,) that contained mostly LBM and BBM The membrane populations contained in P, and P, were subsequently separated by centrifugation (85,000 x g for 14-16 hr) in linear sorbitol gradients (25-606 w/v) Fractions (2 ml) were collected from the top of the gradient and assayed for protein (Coomassie blue dye binding using reagents obtained from Bio-Rad), marker enzymes (aryl esterase for ER-Golgi; K-stimulated phosphatase for LBM; sucrase for BBM), and for NPY binding In other experiments, all the membranes contained in the whole homogenate were subjected to calcium precipitation to increase the yield of LBM Binding of NPY to Membrane Fractions Aliquots (200 pl) of the gradient fractions derived from the P, pellet were incubated for I hr at room temperature with trace amounts (10-50 pM) of labelled NPY dissolved in 800 p1 of 10 mM NaH,PO,-K,HPO,, 0.2 TIUlml aprotinin, 0.5% bovine serum albumin (BSA), 0.05% bacitracin, pH 7.0 When NPY was labelled with lactoperoxidase or iodogen, Triton X-100 was added to the incubation buffer at a final concentration of 0.006% to minimize the nonspecific binding of labelled peptide to the polypropylene assay tubes At the end of the incubation period, the membrane-bound ligand was separated by centrifugation at 27,500 x g for 30 at 4°C Total radioactivity at the beginning of the experiment and in the membrane pellet was determined and specific binding corrected for nonspecific binding (number of apparently bound counts observed 50 zyxwvutsrq zyxw z zy zy zy zyxwvu ANNALS NEW YORK ACADEMY OF SCIENCES in the presence of pM unlabelled NPY.) Scatchard analysis was performed using the EBDA/LIGAND program (originally written by P J Munson and D Rodbard and modified by G A McPherson) obtained commercially from BiosoftlElsevier(Milltown, NJ) Cross-Linking of NPY to Intestinal Membranes Membrane fractions were pooled according to their enzyme markers, washed twice, resuspended to a concentration of mg/ml of membrane protein in mM MgCl,, 50 mM NaCl, 0.5 mM EDTA, mM histidine-imidazole, pH 7.4 The membrane pools were frozen in liquid nitrogen, and stored at -70°C Binding studies were performed as in the prior section except that 100 pg of membrane protein was used for each assay The membrane pellet obtained after allowing radiolabelled NPY to bind was washed (centrifugation at 27,500 x g for 30 min) and finely resuspended with a 25 gauge needle in ml of 60 mM HEPES, pH 7.5 Cross-linking was initiated by the addition of 10 p1 of 20 mM DSS dissolved in dimethyl sulfoxide (final concentration -0.2 mM), and the membranes were then incubated for 15 at 4°C on a rotary shaker The crosslinking was stopped by the addition of ml of cold 60 mM HEPES, 60 mM ammonium acetate, pH 7.5 The membranes were centrifuged at 27,500 x g for 30 and washed with 62.5 mM Tris-HC1, pH 6.8, prior to SDS polyacrylamide gel electrophoresis Prepamlion of Bm‘n Membranes, NPY Binding, and Cross-Linking Male Sprague-Dawley rats (200-250 g) were anesthetized with intraperitoneal pentobarbital, decapitated and the brains rapidly removed The cerebellum and white matter were dissected away from the cortex, and the cortex then homogenized with a glasson-glass Dounce homogenizer (6 passes pestle B, 10 passes pestle A) in 10 ml of ice-cold “Buffer A” (137 mM NaCl, 2.68 mM KCl, 2.05 mM MgCl,, mM EDTA, 0.1 mM PMSF, 0.1% bacitracin, 0.1% glucose, 0.2 TIU/ml aprotinin and 20 mM HEPES, pH 7.4) The homogenate was brought to a volume of 30 ml with “Buffer A” and centrifuged at 14.000 x g and 4°C for 15 The resulting pellet was washed and resuspended in a final volume of 4.5 ml buffer A Binding and cross-linking experiments were performed using the same methods described above for the intestinal membrane except that binding was performed in “buffer A” supplemented with 0.005% Triton X-100 and 0.2% BSA and cross-linking was performed with mM DSS SDS-Polyacrykuni& Gel Electrophoresis and Autoradiogmphy Cross-linked membrane pellets were resuspended in 150 p1 of a solubilization buffer containing 2% SDS,10% w/v glycerol, mM EDTA, 10 mM DlT,bromphenol blue and pyronin Y as dye indicators, and 0.0625 M Tris-HC1, pH 6.8; D l T was omitted when using DSP Following incubation in a shaking water bath at 37°C for 30 min, the resulting membrane suspension, or the solubilized material contained in the supernatant after centrifugation at 18,000 x g and 22°C for minutes was studied by 10% polyacrylamide gel electrophoresis and autoradiography as outlined by Nguyen et a1.’5v16 In some experiments, radioactive bands were cut from the dried gels using the autoradiogram as a template and counted directly zyxwvuts zyxwvutsr zy zyxw zyx zyx zyxwvut TAYLOR ef al.: NPY RECEPTOR 51 RESULTS Binding of NPY to Pooled Membrane Fractions zyxwv Specific binding of Bolton-Hunter labelled NPY to LBM (40 pg of membrane protein) was 5.8% ? 1.4% of the total radioactive NPY added Binding of NPY to LBM exhibited dependence upon time and the amount of membrane protein added In contrast the nonspecific binding to BBM observed in the presence of 10 pM NPY was actually higher than the total binding in the absence of unlabelled NPY Half-maximal inhibition of labelled NPY binding to LBM was observed with 20-50 nM unlabelled NPY Scatchard analysis demonstrated equally good fits (P >0.05) for a single binding site model (K, 15 nM, B,, 30 pmolehg of membrane protein) and a two-binding-site model (K, 5.3 nM, B,, pmolehg of membrane protein [Site I]; K, 29 nM, B, 36 pmole/mg of membrane protein [Site 21) When NPY was radiolabeled with lactoperoxidase or IODO-GEN and purified by HPLC, at least major peaks corresponding to different sites of iodination were observed (NPY has tyrosine residues that can potentially be iodinated) Sheikh er af l4 previously demonstrated that, following IODO-GEN radioiodination, NPY contained in the first radioactive peak (IODO-GEN-NPY ,) on HPLC was monoiodinated at tyrosine residue 1; NPY contained in the second radioactive peak (IODO-GEN-NPY,) was monoiodinated at tyrosine residue 36; and NPY contained in the third major peak or radioactivity (IODOGEN-NPY,) was iodinated at both positions and 36 We determined whether the preferential binding of radiolabeled NPY to LBM was dependent on site of iodination of the ligand IODO-GEN-NPY bound well to LBM (11% of the initial amount of NPY added specifically bound to 100 pg of membrane protein) but poorly to BBM (1% specific binding to 100 pg of membrane protein) In contrast, IODO-GEN-NPY, and IODO-GEN-NPY, both bound well to LBM (20-25% specific binding to 100 pg of membrane protein), but also BBM (8-10% of the initial amount of NPY bound specifically to 100 pg of membrane protein) The lactoperoxidaselabeled NPY obtained from the first peak of radioactivity observed on HPLC (lactoNPY was assessed in the same manner The specific binding of NPY to LBM and BBM (75 pg of membrane protein) was respectively 14.8% 1.3% and 1.3% ? 0.1% of the initial amount of radioactive NPY I added Since IODO-GEN-NPY, and IODO-GENNPY, are both radioiodinated at position 36, it is possible that this is the common feature that allows demonstration of ligand binding to BBM , , Assessment of Label Degradation As mentioned above, NPY radioiodinated either at the fourth lysine residue using the Bolton-Hunter agent (B-H-NPY,) or at the first tyrosine residue (NPY,) using either IODO-GEN or lactoperoxidase binds specifically to LBM but not to BBM On the other hand, NPY radioiodinated at the carboxyl terminal tyrosine residue in position 36 (NPY,,) while still exhibiting preferential binding to LBM also exhibited specific binding to BBM This observation raises the possibility that there are receptors on the BBM which are only readily demonstrable with NPY labelled at the carboxyl terminus and not with amino-terminal labelled NPY (NPY,) When the putative NPY receptors were identified by cross-linking to radiolabelled NPY no receptor species unique to NPY,, was identified l It is possible that the binding reflects contamination of BBM with LBM However, the degree of binding observed with the carboxyl terminal labelled tracers makes this unlikely Since BBM contain proteases, preferential degradation of NPY and , 52 z zyxwvutsrq ANNALS NEW YORK ACADEMY OF SCIENCES 10 FIGURE Time and temperature dependence of Bolton-Hunter-NPY degradation by BBM B-H-NPY was incubated with BBM (20 pg of membrane protein) for the times noted on the abscissa After the incubation was completed, the suspension was centrifuged at 27,500 x g for 30 The degree of degradation of the unbound radiolabeled NPY contained in the supernatant was assessed by 10% TCA precipitation (30 at 4°C) zyxwvutsrqp zyxwvutsrq - zyxwvutsr , 10 I 20 ( 30 I 40 , 50 I 60 Time (min) BH-NPY relative to NPY,, could account for the apparent differential binding of the different radioligands to BBM To address this question, NPY label degradation was assessed by monitoring either the fraction of radiolabelled NPY that did not precipitate with 10% TCA or the fraction which could not be rebound to LBM In the latter experiments, Bolton-Hunter labelled NPY was incubated for hr at 22"C, either in the absence of membranes or in the presence of 70 pg of either LBM or BBM At the end of the incubation period, the radiolabelled NPY that was not bound to the membranes was recovered after centrifugation (27,500 x g for 30 min) and allowed to rebind to LBM, in the presence or absence of p M unlabelled NPY The specific binding of BH-NPY to LBM was determined as the difference in the amount of radiolabelled NPY bound in the absence or presence of excess unlabelled NPY Compared to the control preincubation without membrane, preincubation with LBM did not alter specific binding to LBM in the second incubation (1 1.5% after preincubation with LBM or without membranes) In contrast, after hr preincubation of BH-NPY with BBM, the subsequent specific rebinding to LBM was only 27% of control (specific binding of 3%) In a similar fashion, after an incubation with BBM, the unbound BHNPY showed a 60% decrease in TCA precipitability (from 87% to 36%) Degradation of BH-NPY by BBM was time and temperature dependent (FIG.1) We next determined whether there was differential degradation of NPY, and NPY,, by BBM by examining degradation of different forms of IODO-GEN radiolabelled NPY (FIG 2A) After 30 incubations of label peaks at 22°C with 40 p g BBM, q e percentages of unbound radiolabelled NPY that could be TCA-precipitated was 51% for peak (NPY,), 92% for peak (NPY,,), and 90% for peak (NPY, a 36) By COmpXison, the corresponding percentages for LBM were , and 94% respectively The greater degradation of NPY, by BBM was little altered by the addition of an excess amount (1 pM) of unlabelled NPY Similar results were obtained when the different forms of lactoperoxidase-labelledNPY (FIG 2B) were studied Thus, after incubation with 50 pg membranes, the fractions of lactoperoxidase-radiolabelledNPY that could be TCA-precipitated was 87% (peak 1) and 95% (peak 2) after a preincubation with LBM, and 40% and 82% after a preincubation with BBM This finding correlates well with the 50% loss in the expected rebinding ability of NPY to LBM after preincubation with BBM In the next series of experiments, we attempted to minimize enzymatic degradation of NPY Even with shorter incubations at 4°C in the presence of a combination of protease inhibitors (1 mM of phenylmethylsulfonylfluoride and 0.0125 mglml each of pepstatin, chymostatin, antipain, leupeptin, trypsin inhibitor), degradation of NPY was not fully inhibited When degradation was partially inhibited, small increases in specific binding of , zyxw z , zyxwvutsr zyxwvuts zyxwvu zy zyxwvu TAYLOR et d.:NPY RECEPTOR 53 NPY I to BBM could be demonstrated, suggesting that degradation accounts at least in part for the lack of NPY binding to BBM However, since the conditions used to limit degradation (shorter incubations at 4°C) resulted in decreased specific binding of all labels, including NPY,,, we cannot assess with certainty whether the lack of NPY, binding to BBM was due entirely to degradation In any case, regardless of the radiolabel used, NPY bound less well to BBM than to LBM, suggesting that degradation will not entirely account for the preferential binding of NPY to LBM Since only the radioactivity of the radiolabeled NPY is monitored, we cannot determine if regions of the molecule other than those containing the labelled tyrosine residue undergo degradation However, these results provide evidence that there is preferential degradation of the amino terminus of NPY by BBM , zyxwvuts zy Cross-Linking the NPY Receptor in Intestine and Brain Intestine Lacto-NPY I was cross-linked to its LBM receptor using DSS and the resulting NPYreceptor complexes analyzed on SDS-PAGE followed by radioautography (FIG.3) The NPY-receptor complexes, which were cross-linked in the absence of the reducing agent DTT (FIG.3; lane 3) migrated as a major radioactive band with an Mr of 52,000-59,000 and as a minor band with an Mr of 42,000-44,OOO (the ranges reflect the apparent molecular weights estimated using the top and bottom of each band, and are derived from eight separate experiments) Radioactivity at the top of the lane represents aggregated material larger than 220 kDa (the size of the largest molecular weight marker, femtin); the FIGURE Differential degradation of radiolabeled NPY by intestinal LBM and BBM: NPY was radiolabeled with either IODO-GEN or lactoperoxidase and the different peaks obtained from HPLC purification incubated with LBM or BBM at 22°C When the incubation was completed, the suspension was centrifuged at 27,500 x g for 30 The degree of degradation of the unbound radiolabeled NPY contained in the supernatant was assessed by 10% TCA precipitation (30 at 4°C) (A) IODOGEN radiolabelled NPY incubation for 30 with 40 pg of membrane protein; (B) lactoperoxidase radiolabeled NPY, incubation for 60 with 50 pg of membrane protein 54 zyxwvutsrqp zy zyxw zyxwvu zyx ANNALS NEW YORK ACADEMY OF SCIENCES activity at the front of the gel is thought to reflect radiolabelled NPY that was bound, but not cross-linked to the membranes, which then became dissociated during SDS treatment Cross-linking was blocked by the addition of 10 +M cold NPY prior to the incubation with DSS (lanes and 4) Incubation of the cross-linked membranes in 10 mM DTT prior to SDS-PAGE (lane 1) minimally affected the migration of the 52-59-kDA band but FIGURE Size difference between intestinal and brain NPY-receptor complexes Intestinal laterobasal membranes (37.5 kg of membrane protein) or crude brain membranes (370pg membrane protein) were incubated with trace amounts of lactoperoxidase radioiodinated NF'Y in the presence or absence of unlabeled 10 pM NPY The NPY-receptor complexes were next cross-linked using 0.2 mM (intestinal) or mM (brain) of DSS and analyzed subsequently by SDS 10% polyacrylamidegel electrophoresis in the presence or absence of the reducing agent D" The resulting radioautograph is shown On the 1eB of the radioautograph, the positions of the molecular weight standards and the bands corresponding to the intestinal (b)or brain (+) NPY receptors are shown zyxwvutsr shifted the position of the 42-44 kDa species to Mr 37-39 kDa This later change may reflect intramoleculardisulfide bonding within the 42-44 kDa species or disulfide bonding between a 37-39 kDa binding subunit and a small kDa subunit Additional faint bands at Mr -75,000-90,000 and -1lO,OOO-l20,OOO were also revealed; these addi- zyxwvuts zyxwvutsrqp zyxwvzy TAYLOR ef al.: NPY RECEPTOR 55 tional bands reflect specific binding of NPY in that they were inhibited in the presence of FM unlabelled NPY Brain When radioactive NPY was cross-linked to the brain receptor with DSS, two radioactive bands were seen (FIG.3: lane 6) which migrated at approximately 62 kDa (range 60-64) and 39 kDa (range 36-39) In contrast to the intestinal receptor the addition of 10 mM D l T did not appreciably alter the migration pattern of either band (lane ) DISCUSSION zyxw zyx NPY is localized to the intrinsic nerves of the small intestine, and may be released from nerve endings to directly inhibit intestinal secretion.*-' ' Interaction between NPY and specific receptors located on the serosal side of the intestinal epithelial cell is a necessary prerequisite for this biologic action Using a membrane preparation technique that allows fractionation of LBM free of ER-Golgi, we have demonstrated that NPY, radiolabelled with the Bolton-Hunter agent or at tyrosine (with IODO-GEN and lactoperoxidase) binds exclusively to intestinal LBM l This finding is consistent with the observation that NPY will only inhibit secretion when applied to the serosal surface of mucosal strips mounted in Ussing chambers In competitive inhibition studies, unlabelled NPY inhibited B-H NPY binding with an IC,, of 20-50 nM Thus the measured affinity of the intestinal receptor was not as high as the values previously described for the brain and kidney NPY receptors, 14*'7-19 or for a PYY-preferring receptor described on crude intestinal membranes." This 4- to 5-fold lower affinity may be partially due to the small degree of ligand degradation by laterobasal membranes Alternatively, the low affinity could reflect greater receptor degradation during the 2-day-long intestinal membrane fractionation, which might occur despite the addition of protease inhibitors at the beginning of the procedure However, it should be noted that this lower K, is still within the range of the EC,, (10-30 nM) estimated for the biologic effects of NPY on the intestine.'-' We have identified two tentative candidates for the LBM NPY receptor after crosslinking the receptor followed by analysis on SDS-PAGE under reducing conditions: a major 52-59 kDA band and a minor 37-39 kDa band In the absence of DTT, the 37-39 kDa band migrates as a 42-44 kDa species This finding suggests the presence of disulfide bonding within the receptor itself or between a 37-39 kDa binding subunit and a separate -5-kDa subunit IODO-GEN-NPY,, IODO-GEN-NPY,, and lacto-NPY, crosslinking to LBM with DSS revealed additional faint bands at 75-90 kDa and 110-120 kDa l These faint bands were also seen with DSP cross-linking of lacto-NPY,, and as such they not appear to be specific for IODO-GEN-NPY, or lacto-NPY, Since these bands were faint and inconsistent, we have had difficulty in further analyzing their relationships to the 52-59 kDa- and 37-39/4244 kDa species Since both the 52-59 and 37-39 kDa species have the same affinity for NPY, we speculate that the smaller product is derived as a result of degradation from the larger species While Inui et al." reported the molecular weight for the NPYlPYY receptor in the brain to be of 50 kDa, we demonstrated two candidate species for the brain NPY receptor, with Mr's of 62,000 and 39,000.23 The slight difference in size between the intestinal 52-59 kDa species and the brain 62 kDa species was confirmed when the cross-linked NPY-receptor complexes from these two different tissues were analyzed together on the ' 56 zy zyxwvutsrq ANNALS NEW YORK ACADEMY OF SCIENCES same gel (FIG 3) Another difference between intestine and brain is that the smaller species in the intestine [37-39/42-44 kDa] is sensitive to reducing agents while the brain 39 kDa species is not Alternate methodologies (e.g., immunologic mapping, receptor purification and amino acid analysis or receptor cloning) will be required to determine whether these slight differences reflect distinct structures, or artifactual modifications of the receptor secondary to enzymatic degradation of the larger species (75-90 kDa or 115-120 kDa) IODO-GEN-NPY, and IODO-GEN-NPY, bound equally well to the brain NPY receptor,14 while IODOGEN-NPY, bound better than IODO-GEN-NPY to LBM and BBM derived from enterocytes The binding of IODO-GEN-NPY, and IODO-GENNPY, coupled with the lack of binding of IODO-GEN-NPY to brush border membranes is intriguing IODO-GEN-NPY is iodinated at tyrosine residue 1, IODO-GEN-NPY, is iodinated at tyrosine residue 36, and IODO-GEN-NPY, is iodinated at both tyrosine residues and 36 The differential binding of the different labels to LBM and BBM could reflect two classes of NPY receptors: a class localized to LBM which can bind B-H-NPY and NPY radioiodinated at the tyrosine residue 1, and an additional class localized to BBM (and possibly LBM) which only recognizes NPY radioiodinated at position 36 However, in our cross-linking experiments, we have been unable to provide structural evidence for distinct NPY receptors which can only bind NPY radiolabelled at position 36 It is conceivable that binding of NPY to BBM reflects contamination with LBM However, the magnitude of binding of NPY,, labels to BBM makes this unlikely as BBM would have to be heavily contaminated Our studies in differential degradation of different label species by LBM and BBM suggest the presence in BBM of an enzyme(s) that cleaves the amino terminus of NPY thereby removing the label in the Tyr, position and the Bolton-Hunter label In summary, we have localized receptors for NPY to the serosal laterobasal membrane of the intestinal epithelial cell By covalently cross-linking NPY to its receptor, we have demonstrated two main molecular species of the receptor with molecular sizes of 48-55 kDa (52-59 kDa minus molecular weight of NPY) and 33-37 kDa While NPY iodinated at Tyrosine, is extensively degraded by BBM we have observed that NPY iodinated at Tyrosine,, will bind to BBM zyx , , , SUMMARY Neuropeptide Y (NPY) is widely distributed in the central and peripheral nervous systems where it serves neuromodulator and neurotransmitter functions NPY is contained within intrinsic nerves of the small intestine and can be demonstrated to inhibit intestinal secretion when added to the serosal side of intestine mucosa mounted in Ussing chambers When injected centrally it has potent effects on food intake, blood pressure, sexual activity and circadian rhythms Using NPY radiolabeled with iodogen, lactoperoxidase, or the Bolton-Hunter reagent, we have localized high-affinity NPY receptors on brain membranes and on the serosal laterobasal membranes of the rat intestinal epithelial cell We have demonstrated that enzymatic degradation may limit the ability to demonstrate NPY binding to brush border membranes In other experiments NPY was cross-linked to its receptors in brain and intestine using disuccinimido suberate and the resulting complexes analyzed on SDS polyacrylamide gel electrophoresis followed by radioautography We identified two main NPY receptor species in the intestine with molecular sizes of 52-59 kDa and 37-39 kDa The 37-39 kDa species may possess a disulfide bond which gives the receptor a fixed conformation, or it may be composed of two subunits (37-39 kDa and -5 kDa subunits) This conclusion is based on the different migration of the zyxwvuts zyxwvuts zyxwv zy TAYLOR et ul.: NPY RECEPTOR 57 zyxwv smaller band in the presence of the reducing agent, dithiothreitol The intestinal NPY receptor exhibits differences from the rat brain receptor previously characterized by us using similar techniques The brain receptor has a molecular weight of approximately 58 kDa with a smaller species of about 35 kDa which shows no differences in migration after exposure to dithiothreitol The localization of NPY receptors on laterobasal membranes and brain membranes is consistent with previous anatomic and physiologic findings The different characteristics of each receptor type provides physical evidence of receptor heterogeneity However, it is possible that the greater enzymatic degradation observed in intestinal membranes might explain the differences in receptor sizes in the two organs ACKNOWLEDGMENT The authors are indebted to Dr Steven R Vigna for his help with the radioiodination of NPY zyxwv REFERENCES I TATEMOTO, K 1982 Roc Natl Acad Sci USA 79: 2514-2518 TATEMOTO, & V MUTT.1982 Nature 2%: 659-660 K., M CARLQUIST TATEMOTO, K 1983 Biomedical Res 4(Suppl.): 1-6 KIMMEL, J R., L 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NGUYEN.1989 Am J Physiol 18 MANNON,P J., I L TAYLOR, 256: G637-643 M., C M M MILES,K LEYS& P S SEVER 1987 J Cardiovasc Pharmacol 19 SCHACHTER, lO(Supp1 12) S157-SI62 M., B CHENUT,C ROUYER-FESSARD, K TATEMOTO, A COUVINEAU, A 20 LABURTHE, SERVIN & B AMIRANOFF 1986 Endocrinology 118 1910-1917 T INOUE, N SAKATANI M OYA,H MORIOKA, K SHII,K YOKONO, 21 INUI,A., M OKITA, N MIZUNO& S BABA.1989 Endocrinology 124: 402-409 ... terminus of NPY by BBM , zyxwvuts zy Cross-Linking the NPY Receptor in Intestine and Brain Intestine Lacto-NPY I was cross-linked to its LBM receptor using DSS and the resulting NPYreceptor complexes... specific binding of NPY in that they were inhibited in the presence of FM unlabelled NPY Brain When radioactive NPY was cross-linked to the brain receptor with DSS, two radioactive bands were... unlabelled NPY inhibited B- H NPY binding with an IC,, of 20-50 nM Thus the measured affinity of the intestinal receptor was not as high as the values previously described for the brain and kidney