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 ob
Trang 1Comparison of the Neuropeptide
IAN L TAYLOR, PETER J MANNON, GREGORY G HEINTZ,
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 neuromod-
ulator functions in the central, peripheral and enteric nervous Central admin-
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-to-
serosal Na+ and C1- fluxes, and reduces serosal-to-mucosal C1- fluxes.*-" This inhib-
itory 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 mem-
branes (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
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 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA40506) and Dr Ian Taylor of a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAR01 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-ethanesulfonic acid;
BSA: bovine serum albumin; DlT: dithiothreitol; SDS: sodium dodecyl sulfate; PMSF: phenylme-
thylsulfonyl fluoride; DSS: disuccinimido suberate; EDTA: ethylenediaminetetraacetic acid; PAGE:
polyacrylamide gel electrophoresis; lacto-NPY ,: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAlactoperoxidase 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
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METHODS
Chemicals and Reagents zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Synthetic NPY, PYY and PP were urchased from Peninsula Laboratories (Belmont,
gland Nuclear (specific activity 2200 Cilrnmol) For some experiments one or more of the
five tyrosines in NPY was radioiodinated using lactoperoxida~e'~ or IODO-GEN, l 4 and
the resulting radiolabelled species purified on high performance chromatogra h
from Amersham C o p (Arlington Heights, IL), polyacrylamide gel electrophoresis re-
agents from Bio-Rad (Richmond, CA) and molecular weight standards for gel electro-
phoresis from Pharmacia (Pistcataway, NJ) Triton-X-100 was from J.T Baker (Phil-
lipsburg, NJ), and all the protease inhibitors (antipain, chymostatin, pepstatin, leupeptin
phenylmethylsufonyl fluoride (PMSF), ovomucoid trypsin inhibitor, bacitracin, aproti-
nin) were from Sigma The crosslinking agent disuccinimido suberate (DSS) was pur-
chased from Pierce (Rockford, IL) X-ray film (XAR-2) was from Eastman Kodak (Roch-
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 1 mM
PMSF) and during the first homogenization (25 kglml each of antipain, chymostatin,
pepstatin, leupeptin, ovomucoid trypsin inhibitor, and 1 mM PMSF) Differential cen-
trifugation was used to yield a pellet (P,) that contained mostly ER-Golgi and LBM;
precipitation of ER-Golgi with 8 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 phos-
phatase 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 sep-
arated by centrifugation at 27,500 x g for 30 min 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
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in the presence of 1 pM unlabelled NPY.) Scatchard analysis was performed using the
EBDA/LIGAND program (originally written by P J Munson and D Rodbard and mod-
Cross-Linking of NPY to Intestinal Membranes
Membrane fractions were pooled according to their enzyme markers, washed twice,
NaCl, 0.5 mM EDTA, 5 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 (centrif-
ugation at 27,500 x g for 30 min) and finely resuspended with a 25 gauge needle in 1 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 min at 4°C on a rotary shaker The crosslinking was stopped
by the addition of 2 ml of cold 60 mM HEPES, 60 mM ammonium acetate, pH 7.5 The
membranes were centrifuged at 27,500 x g for 30 min and washed with 62.5 mM
Tris-HC1, pH 6.8, prior to SDS polyacrylamide gel electrophoresis
Prepamlion zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Bm‘n Membranes, NPY Binding, and Cross-Linking
Male Sprague-Dawley rats (200-250 g) were anesthetized with intraperitoneal pen-
tobarbital, decapitated and the brains rapidly removed The cerebellum and white matter
were dissected away from the cortex, and the cortex then homogenized with a glass-
on-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,, 1 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 min 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 1 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, 1 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 5 minutes was studied by 10% polyacrylamide
gel electrophoresis and autoradiography as outlined by Nguyen et a1.’5v16 In some ex-
periments, radioactive bands were cut from the dried gels using the autoradiogram as a
template and counted directly
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RESULTS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA51 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Binding of NPY to Pooled Membrane Fractions zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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 Scat-
(K, 15 nM, B,,, 30 p m o l e h g of membrane protein) and a two-binding-site model (K,
5.3 nM, B,,, 4 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 3 major peaks corresponding to different sites of iodination were observed
(NPY has 5 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 (IODO-
GEN-NPY,) was iodinated at both positions 1 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 p g of membrane
protein) but poorly to BBM (1% specific binding to 100 p g 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 lactoperoxidase-
labeled NPY obtained from the first peak of radioactivity observed on HPLC (lacto-
NPY was assessed in the same manner The specific binding of NPY , to LBM and BBM
(75 pg of membrane protein) was respectively 14.8% 2 1.3% and 1.3% ? 0.1% of the
initial amount of radioactive NPY I added Since IODO-GEN-NPY, and IODO-GEN-
NPY, 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 6 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
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FIGURE 1 Time and temperature depen- dence 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 com- pleted, the suspension was centrifuged at 27,500 x g for 30 min The degree of degrada- tion of the unbound radiolabeled NPY con- tained in the supernatant was assessed by 10%
TCA precipitation (30 min at 4°C)
0 , I ( I , I
Time (min)
BH-NPY zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBArelative 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 1 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 1 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 1 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 BH-
NPY 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 min 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
ison, the corresponding percentages for LBM were 8 9 8 , 9 6 8 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-labelled NPY (FIG 2B) were studied Thus, after incubation
with 50 p g membranes, the fractions of lactoperoxidase-radiolabelled NPY 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
peak 1 (NPY,), 92% for peak 2 (NPY,,), and 90% for zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBApeak 3 (NPY, a 36) By COmpX-
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NPY I to BBM could be demonstrated, suggesting that degradation accounts at least in
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
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 NPY-
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 2 Differential degradation of radio- labeled NPY by intestinal LBM and BBM:
NPY was radiolabeled with either IODO-GEN
or lactoperoxidase and the different peaks ob- tained 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 min The degree of deg- radation of the unbound radiolabeled NPY con- tained in the supernatant was assessed by 10%
TCA precipitation (30 min at 4°C) (A) IODO-
GEN radiolabelled NPY incubation for 30 min with 40 pg of membrane protein; (B) lactoper- oxidase radiolabeled NPY, incubation for 60
min with 50 pg of membrane protein
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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
to SDS-PAGE (lane 1) minimally affected the migration of the 52-59-kDA band but
FIGURE 3 Size difference between intestinal and brain NPY-receptor complexes Intestinal lat-
erobasal membranes (37.5 kg of membrane protein) or crude brain membranes (370 pg 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 1 mM (brain) of DSS and analyzed subsequently by SDS 10% polyacrylamide gel
electrophoresis in the presence or absence of the reducing agent D" The resulting radioautograph
is shown On the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1eB of the radioautograph, the positions of the molecular weight standards and the
bands corresponding to the intestinal (b) or brain (+) NPY receptors are shown
shifted the position of the 42-44 kDa species to Mr 37-39 kDa This later change may
reflect intramolecular disulfide bonding within the 42-44 kDa species or disulfide bond-
ing between a 37-39 kDa binding subunit and a small 5 kDa subunit Additional faint
bands at Mr -75,000-90,000 and -1lO,OOO-l20,OOO were also revealed; these addi-
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tional bands reflect specific binding of NPY in that they were inhibited in the presence of
1 FM unlabelled NPY
Brain
When radioactive NPY was cross-linked to the brain receptor with DSS, two radio-
active 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 5 )
DISCUSSION
NPY is localized to the intrinsic nerves of the small intestine, and may be released
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 1 (with IODO-GEN and lac-
toperoxidase) binds exclusively to intestinal LBM l 6 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
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 begin-
ning 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 cross-
linking 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 di-
sulfide 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, cross-
linking to LBM with DSS revealed additional faint bands at 75-90 kDa and 110-120
kDa l 6 These faint bands were also seen with DSP cross-linking of lacto-NPY,, and as
such they do 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
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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
BBM derived from enterocytes The binding of IODO-GEN-NPY, and IODO-GEN-
NPY, 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 1 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
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 com-
plexes 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
Trang 10TAYLOR zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAul.: NPY RECEPTOR zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA57 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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
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
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