Tài liệu Báo cáo Y học: A pool of Y2 neuropeptide Y receptors activated by modifiers of membrane sulfhydryl or cholesterol balance pot

PRIORITY PAPER
A pool of Y2 neuropeptide Y receptors activated by modifiers
of membrane sulfhydryl or cholesterol balance
Steven L. Parker
1
, Michael S. Parker
2
, Justin K. Kane
1
and Magnus M. Berglund
3
1
Department of Pharmacology, University of Tennessee College of Medicine, Memphis, TN, USA;
2
Department of Microbiology
and Molecular Cell Sciences, University of Memphis, TN, USA;
3
Unit of Pharmacology, Department of Neuroscience,
University of Uppsala, Sweden
The cloned guinea-pig Y2 neuropeptide Y (NPY) receptors
expressed in Chinese hamster ovary (CHO) cells, as well as
the Y2 receptors natively expressed in rat forebrain, are
distributed in two populations. A smaller population that is
readily accessed by agonist peptides on the surface of intact
cells constitutes less than 30% of Y2 receptors detected in
particulates after cell homogenization. A much larger frac-
tion of cell surface Y2 sites can be activated by sulfhydryl
modifiers. A fast and large activation of these masked or
cryptic sites could be obtained with membrane-permeating,
vicinal cysteine-bridging arsenical phenylarsine oxide. A
lower activation is effected by N-ethylmaleimide, an alkyla-

tor that slowly penetrates lipid bilayers. The restricted-access
alkylator, 2-[(trimethylammonium)ethyl]methanethiosulfo-
nate, was not effective in unmasking these sites. Some of the
hidden cell surface Y2 sites could be activated by polyene
filipin III through complexing of membrane cholesterol. The
results are consistent with the presence of a large Y2 reserve
in a compartment that can be accessed by alteration of
sulfhydryl balance or fluidity of the cell membrane, and
by treatments that affect the anchoring and aggregation of
membrane proteins.
Keywords: receptor sequestration; receptor reserve; receptor
signaling; receptor masking.
Synaptic discharge of many neurotransmitters produces
concentrations of these receptor agonists that saturate the
respective binding sites, with a potential for prolonged and
excessive signaling. With receptors characterized by high
binding affinities, which represent a large fraction of
rhodopsin-related neurotransmitter receptors, it may not
be possible to adequately constrain the signaling by
dissociation of the agonist. For neuropeptide receptors, a
paracrine regulation via secretion of specific peptidases
would meet large difficulties in both the selectivity and the
economy of action. Scavenging by cell membrane- resident
ectoproteinases by way of in situ encounters with extra-
cellular agonists may not satisfy the clearance needs
created by agonist discharge. A much more selective (and
potentially quicker) regulation could be provided by
sequestration or internalization of the receptor–ligand
complex and further intramembrane or intracellular
processing (reviewed in [1,2]). This could be accomplished

by recycling sequestration (e.g. the m1 muscarinic receptor
[3]), by recycling internalization (e.g. the m2 muscarinic
receptor [4] or the neuropeptide Y (NPY) Y1 receptor [5]),
and by lysosome-linked disposing internalization (e.g. the
endothelin-B receptor [6]), all possibly enacted in relation
to the prevailing levels of the respective agonists and the
extent of preservation of the respective receptor molecules.
Among neuropeptide transmitters, large levels of NPY are
present in many areas of the forebrain [7], enabling an
important regulation of feeding [8]. The forebrain NPY
receptors include all principal Y receptor types [9], with
Y1 and Y2 receptors detected at largest levels [10]. The
slowly internalizing Y5 receptors [11] could represent a
substantial component of sustained feeding regulation by
NPY. However, both the Y5 and the feeding-coregulative
[8] Y1 receptors (which could be strongly driven to
internalize even by picomolar concentrations of NPY
[5,12]) might be overwhelmed by large NPY release, in
view of high nanomolar levels of the peptide in rodent [7]
and even in human forebrain locations [13]. The discharge
overloads could be handled through participation of
another NPY receptor, the Y2 receptor. The Y2 receptor
is strongly expressed especially in hypothalamic areas [10],
and exists in two affinity states, one of which shows a very
high binding affinity and is linked to a large degree of
receptor aggregation [14]. The Y2 receptor is also
distinguished by a low rate of internalization compared
to the Y1 receptor when expressed in CHO cells [12]. A
large portion of the Y2 complement is not detected on
membranes of intact cells, but becomes accessible to

agonist peptides upon cell homogenization, or upon
treatment with a membrane-penetrating crosslinker of
Correspondence to S. L. Parker, Department of Pharmacology,
University of Tennessee College of Medicine, Memphis,
TN 38163, USA.
Tel.: + 1 901 850 7617,
E-mail:
Abbreviations: NPY, neuropeptide Y; hNPY, human/rat NPY;
hPYY(3–36), human peptide YY(3–36); PYY, peptide YY; NEM,
N-ethylmaleimide; MTSET, 2-[(trimethylammonium)ethyl]methane-
thiosulfonate bromide; PAO, phenylarsine oxide.
(Received 22 February 2002, revised 18 March 2002,
accepted 22 March 2002)
Eur. J. Biochem. 269, 2315–2322 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02903.x
vicinal cysteines, phenylarsine oxide (PAO) [12]. This
study presents evidence for activation of these sites by
agents that affect the membrane sulfhydryl balance or
cholesterol, and also the rate of internalization of the Y2
receptor.
MATERIALS AND METHODS
Chemicals
The Y peptides hPYY(3–36) and hNPY were obtained from
the American Peptide Company (San Diego, CA, USA).
Filipin III, N-ethylmaleimide (NEM) and phenylarsine
oxide were purchased from Sigma (St Louis, MO, USA).
Cholesteryl hemisuccinate was obtained from Calbiochem
(La Jolla, CA, USA). Filipin III and PAO were dissolved in
dimethylsulfoxide and stored in aliquots at )80 °C. Filipin
complex (Sigma; a mixture of three isomers of filipin) was
only about 25% as active as filipin III, and hence was not

used. Cholesteryl hemisuccinate was prepared as a water
emulsionandalsostoredat)80 °C. Restricted-access
methanethiosulfonate MTSET (2-[(trimethylammo-
nium)ethyl]methanethiosulfonate bromide) was obtained
from Toronto Research Chemicals (North York, Ontario,
Canada) Solutions of this agent and of NEM were made
within 15 min before use.
Labeled peptides
All iodinations of Y peptides were performed as described
previously [15]. The radioactive Y peptides were 75–90%
monoiodinated and had specific activities in the range of
1500–1800 CiÆmmol
)1
(70–80% theoretical), as deduced by
comparison in saturation assays with HPLC-purified
monoiodinated
125
I-labeled Y peptides hNPY and
hPYY(3–36) supplied by PerkinElmer/NEN, Cambridge,
MA, USA (specific activity 2170 CiÆmmol
)1
).
Cell cultures and labeling
All cell types were cultured in F12/D-MEM medium (Gibco,
Long Island, NY, USA) at 250 lgÆmL
)1
of geneticin and
2m
M
GlutaMax1 (Gibco). The guinea-pig Y1 receptor

(gpY1-CHO; [16]); and the guinea-pig Y2 receptor (gpY2-
CHO; [17]) were expressed in Chinese hamster ovary (CHO)
cells. The cell lines used in this study had stable particulate
receptor density, at the level of 10–20 fmol per 100 000 cells,
over up to 40 passages in F12/D-MEM at 250 lgÆmL
)1
of
geneticin (Gibco). At full confluence, which was reached
within 48 h with seeds of 25 000 cells per cm
2
at 37 °C
in 95% O
2
/5% CO
2
, the cell count was 200 000–235 000
per cm
2
. After four washes with OptiMemÒ medium
(Gibco, Long Island, NY, USA) to remove any external
proteinase activity, the experimental incubations were car-
ried in 48-well (0.8 cm
2
Æwell
)1
) plates, in a volume of
0.25 mL, using OptiMemÒ medium. The labeling of Y
peptides was carried out at 50 p
M
of

125
I-labeled peptide
tyrosine, using 300 n
M
nonlabeled peptides for nonsaturat-
ing (or nonspecific) binding correction. In all experiments,
less than 15% of the labeled peptides were degraded over the
incubation period, as verified by Bio-Gel P-4 chromatogra-
phy [15]. The incubations were terminated by the removal of
the medium by suction, two washes with cold Opti-Mem
buffer, and extraction for 12 min at 0–4 °C with ice-cold
0.2
M
CH
3
COOH/0.5
M
NaCl (pH 2.6), which was found to
quantitatively dissociate the cell-surface attached Y peptides,
without significant extraction of internalized peptides
[12,18]. The binding of Y peptides to particulates from
gpY2-CHO cell or rat forebrain tissue particulates was, on
the other hand, more than 90% extracted by cold acid saline,
as expected from the known importance of the arginine
residues of NPY in Y2 (and Y1) receptor binding [19].
Rat forebrain tissue (a pool of hypothalamic and piriform
cortex slices) was diced by scissors into fragments  1-mm
in diameter, and then dispersed in 0.14
M
NaCl/0.01

M
Na
phosphate/0.001
M
EDTA (NaCl/P
i
/EDTA buffer; final
pH 7.4) at about 50 mg fresh tissue per ml, using five slow
passages through a 12-gauge needle. The suspension was
brought to 5 FALGPA units per mL (5 lmol of furyl-
acroyl-Leu-Gly-Pro-Ala hydrolyzed per min at pH 7.5 and
25 °C) of Sigma collagenase and incubated for 60 min at
23–24 °C with five slow passages through 16-gauge and 18-
gauge needles repeated at 10-min intervals. The suspension
was then filtered through a 100-mesh sieve, and the filtrate
sedimented for 5 min at 100 g to recover the cells. The cells
(>95% excluding Trypan blue stain) were resuspended in
Opti-Mem buffer, and  200 000 cells per well were imme-
diately plated for experimental incubations. The density of
Y2 receptors did not significantly change in the above
dissociation procedure and the additional incubation of up
to 2 h at 37 °C. At the end of the incubation period, the cells
were brought to 0–4 °C, harvested by sedimentation at
100 g for 5 min and surface-washed with cold Opti-Mem
buffer. The pellets were then dispersed in ice-cold 0.2
M
CH
3
COOH/0.5
M

NaCl, and after 12 min at 0–4 °C
sedimented for 5 min at 4000 g to separate the extracted
(originally cell-surface attached) and the residual (internal-
ized) radioactive peptide.
Receptor characterization
The homogenization or NPY receptor assay buffer con-
tained 8% sucrose, 0.2% proteinase-free BSA (Sigma),
0.025% bacitracin, 1 m
M
diisopropylfluorophosphate
(Sigma), 4 m
M
CaCl
2
,2m
M
MgCl
2
,20m
M
hepes.NaOH
(pH 7.4) and 50 l
M
ATP. Particulates from gpY2-CHO
cells or from dispersed rat forebrain cells were isolated by
homogenization in the cold NPY receptor assay buffer,
applying 12 complete strokes of a Teflon pestle (clearance
0.10 mm) in a Potter-Elvehjem homogenizer at about
800 r.p.m., followed by the removal of debris for 5 min at
100 g, and the sedimentation of particulates for 15 min at

16 000 g (all at 0–4 °C). The pelleted particulates were
aliquoted and kept at )80 °C. Particulate receptors were
assayed as described previously [20]. The particle concen-
tration was 100–125 lgÆmL
)1
, the assay volume was
0.2 mL, and the incubation time was 90 min at 23–24 °C,
with the appropriate competitors or inhibitors. The assay
was terminated by centrifugation for 15 min at 12 000 g at
4 °C, the supernatants were discarded, and the pellets
surface-washed by cold assay buffer prior to counting in a
gamma-scintillation counter. The binding properties of the
cell-surface receptors were characterized on monolayer
cultures in Opti-Mem medium. Iodinated Y peptides were
input at 50 p
M
, and competed by up to nine concentrations
of homologous or isologous peptides in the range of
2316 S. L. Parker et al. (Eur. J. Biochem. 269) Ó FEBS 2002
3 · 10
)11
to 1 · 10
)6
M
. Polyethyleneglycol precipitation of
particulates was carried out as described previously [15].
Data evaluation
Binding parameter calculations were carried out in the
LIGAND
program [21]. Multiple comparisons following a

positive
ANOVA
were done in Tukey’s t-test [22].
RESULTS
Activation of cell surface Y2 sites by phenylarsine oxide
PAO inhibited the binding of Y2-selective agonist hPYY
(3–36) to particulates from gpY2-expressing cells or rat
forebrain only at concentrations above 1 m
M
(Table 1).
Pretreatment with the arsenical at 100 l
M
decreased the
affinity of the Y2 binding to CHO cell or forebrain
particulates by not more than 40% (Table 2). However,
PAO consistently increased the Y2 agonist binding to CHO
cell monolayers by fourfold to fivefold (Fig. 1A), as
previously shown [12]. This activation occurred with little
change in Y2 site affinity (Fig. 1A). The binding of the Y2
agonist to dispersed rat forebrain cells was also increased by
PAO up to fourfold (Fig. 1B), without a significant change
in affinity (Fig. 1B). The activation by PAO saturated with
increasing concentration of the arsenical between 10 and
30 l
M
with either the Y2-CHO cells (Fig. 1A), or with rat
forebrain cells (Fig. 1B). Pretreatment of gpY1-CHO cells
at 24 or 37 °C with 30 l
M
PAO did not produce significant

change in subsequent surface labeling by
125
I-labeled hNPY
or
125
I-labeled (Leu31,Pro34)hPYY (data not shown).
The activation of Y2 sites on intact cells by PAO did not
involve more than half of the receptors detectable in
particulates after cell homogenization (Fig. 1A). Homoge-
nization of gpY2-CHO cells alone resulted in a large
activation of Y2 sites, to about twice the level measured
after treatment of cell monolayers by PAO, and exceeding
the monolayer control binding by a factor of at least four in
the absence of PAO treatment. Pretreatment or cotreatment
of either the cell monolayers or the isolated particulates by
PAO (at 30 l
M
) did not produce a consistent increase in
particulate Y2 binding relative to control preparations
(Fig. 1A). Likewise, the Y2 binding to particulates isolated
from homogenates of dispersed rat forebrain cells was
increased more than sixfold relative to whole cells, but not
Table 1. Inhibition of Y2 receptor binding by the agents used. Particu-
lates from gpY2-CHO cells or rat piriform cortex were labeled over
90 min at 23–24 °C by 50 p
M
125
I-labeled hPYY(3–36) in the presence
of 10–12 different concentrations of the respective agents, between 0.01
and 10 m

M
. The results are averages of three separate experiments,
shown ± SEM. The assay conditions are specified in the Methods
section. The nonspecific binding was defined at 300 n
M
of nonlabeled
hPYY(3–36). Percent of the total specific binding displaced at 10 m
M
of an agent is shown in parenthesis after the corresponding K
I
value.
K
I
,m
M
Agent guinea pig Y2-CHO rat forebrain
Dithiothreitol 3.37 ± 0.66 (46%) 3.35 ± 0.44 (34%)
Phenylarsine oxide 2.24 ± 0.75 (78%) 1.51 ± 0.34 (94%)
N-Ethylmaleimide 0.146 ± 0.014 (75%) 0.708 ± 0.104 (73%)
MTSET 0.264 ± 0.027 (72%) 1.86 ± 0.14 (65%)
Filipin III > 0.1 > 0.1
Table 2. Effect of pretreatment with various sulfhydryl-active agents on
theaffinityofY2binding. The K
d
values are in p
M
hPYY(3–36),
± SEM. The particulates were preincubated for 30 min at 24 °C in the
assay buffer in presence of the indicated molarities of SH-active agents,
then sedimented to remove the agents, surface-washed and assayed (see

Materials and methods). Data represent triplicate competition assays
employing 8–10 concentration points in the range of 10–30 000 p
M
nonlabeled Y2 agonist, and 50 p
M
125
I-labeled hPYY(3–36). Defining
the nonspecific binding at 100 n
M
nonlabeled hPYY(3–36), more than
90% displacement of
125
I-labeled hPYY(3–36) was observed at 30 n
M
unlabeled hPYY(3–36) after any treatment. Note that the Y2 binding
inbothCHOandratforebraincellscouldberesolvedintotwosig-
nificant components with K
d
values of 5–15 and 300–700 p
M
,
respectively [14].
K
d
,p
M
Agent and molarity used gpY2-CHO cells Rat forebrain cells
Control 422 ± 40 316 ± 43
Dithiothreitol (1 m
M

) 541 ± 70
Phenylarsine oxide (100 l
M
) 627 ± 70 334 ± 52
N-Ethylmaleimide (30 l
M
) 601 ± 72
MTSET (100 l
M
) 637 ± 46
Fig. 1. Effects of phenylarsine oxide (PAO) on the binding of Y2 agonist
125
I-labeled hPYY (3–36) to gpY2-CHO monolayers or particulates, and
to dispersed rat forebrain cells. The labeled Y2 agonist was input at
50 p
M
in all cases. For assay details see Methods. All data, shown
± SEM, are averages of three experiments. Asterisks indicate differ-
ences significant vs. the control binding in the Tukey t-test at the level
of 95% (*) or 99% (**) confidence. (A) The surface binding to
monolayers or particulates from gpY2-CHO cells as related to pre-
treatment (30 min at 37 °C) of monolayers with PAO in the range of
3–100 l
M
, and pretreatment or cotreatment of particulates with 30 l
M
of the arsenical. Competition by nonlabeled hPYY(3–36) showed K
d
of 442 ± 37 p
M

for control surface binding, and of 638 ± 74 p
M
for
surface binding after pretreatment by 30 l
M
PAO (n ¼ 3). (B) The
surface binding to dispersed rat forebrain cells after pretreatment
(30 min at 37 °C) with 3–100 l
M
PAO,andalsothebindingtopar-
ticulates from cells pretreated for 30 min at 37 °C with 30 l
M
PAO.
Competition by nonlabeled hPYY(3–36) showed K
d
of 407 ± 38 p
M
for control surface binding, and of 451 ± 24 p
M
for surface binding
after pretreatment by 30 l
M
PAO (n ¼ 3).
Ó FEBS 2002 Masked Y2 NPY receptors (Eur. J. Biochem. 269) 2317
further augmented significantly by a pretreatment with
30 l
M
PAO (Fig. 1B).
Theeffectof30l
M

PAO could be largely suppressed by
100 l
M
sulfhydryl protector dithiothreitol, and was com-
pletely prevented by 1 m
M
dithiothreitol (Fig. 2). Dithio-
threitol reduced the binding of hPYY(3–36) to a fraction
(<50%) of particulate Y2 sites at a K
I
value of about
3.5 m
M
with either the gpY2 or the rat forebrain Y2
receptor (Table 1). However, dithiothreitol did not affect
the binding to cell surface sites at up to 1 m
M
(Fig. 2).
Pretreatment with dithiothreitol at up to 1 m
M
did not
affect gpY2 internalization relative to controls, and also
neutralized any decrease in receptor- linked Y2 ligand
internalization by PAO (Fig. 2).
Effects of the alkylators NEM and MTSET
Effects of alkylators on availability of Y2 sites were
examined only with gpY2 receptor expressed in CHO cells.
NEM, an alkylator slowly penetrating the lipid bilayer [23],
also significantly activated the monolayer Y2 sites. The
increase appeared to saturate between 10 and 100 l

M
(Fig. 3), and was followed by a significant decrease at
300 l
M
, reflecting inactivation of a fraction of the Y2 sites at
high concentrations of the alkylator [14]. The restricted-
access alkylator MTSET [24] did not produce a significant
stimulation of the gpY2 surface binding at 10–100 l
M
,and
was inhibitory at 300 l
M
(Fig. 3). Both alkylators essen-
tially prevented internalization of hPYY(3–36) at 300 l
M
,
and their activity at that concentration was fully neutralized
by 1 m
M
dithiothreitol (Fig. 3); a highly selective blockade
of internalization by NEM was also found for the gpY1-
CHO receptor (data not shown).
Activation of Y2 sites by filipin III
Possible effects of the cholesterol-complexing polyene filipin
III on the availability of cell-surface Y2 receptors were also
assessed in gpY2-CHO cells. A substantial increase in
surface Y2 binding could be shown, dependent on concen-
tration of the antibiotic. The increase reached about twice
the control level at 3 l
M

was somewhat reduced at 10 l
M
,
and then dropped to almost the control levels at 30 l
M
(Fig. 4). In the same set of experiments, the increase in
surface Y2 binding at 10 l
M
PAO was, as routinely
observed, close to five times the control value (Fig. 4).
Stimulation of the binding by filipin was largely prevented
by equimolar cholesterol, and was essentially cancelled at a
cholesterol concentration three times in excess to that of
filipin (Fig. 4). The unmasking of the Y2 sites by 10 l
M
PAO was not altered by equimolar cholesterol (Fig. 4).
Cholesterol alone at 10 l
M
slightly increased the surface
binding of the Y2 agonist (Fig. 4).
Activation of the Y2 sites by nonionic detergents or
emulsifiers could not be satisfactorily studied with cell
monolayers, due to loss of cell attachment. With either
gpY2-CHO or rat forebrain particulates, pretreatment with
polyoxyethylene sorbitan emulsifiers Tween 40 (monopalm-
itate) or Tween 80 (monooleate) at up to 10 m
M
produced
less than 10% activation (data not shown).
Dynamics of activation of the surface Y2 sites

by phenylarsine oxide indicates little accumulation
due to receptor externalization
The dynamics of appearance of additional surface sites at
30 l
M
PAO indicated a fast activation, as the increase in the
labeling by agonist peptides relative to control values
Fig. 2. Unmasking of the binding sites for Y2 selective ligand
125
I-labeled hPYY (3–36) on gpY2-CHO cells by phenylarsine oxide is
prevented by dithiothreitol. The cells were pretreated for 30 min at
37 °C with 30 l
M
PAO with or without dithiothreitol (100 l
M
)1m
M
),
or with 1 m
M
dithiothreitol alone, washed and labeled with
125
I-labeled
hPYY(3–36) for 30 min at 37 °C,followedbyextractionofsurface-
bound ligand with acid saline at 0–4 °C (see Materials and methods).
The data are averages of three experiments. In this and further graphs,
asterisks indicate differences significant vs. the control cell surface
binding at the level of 95% (*) or 99% (**) confidence in Tukey t-tests,
while ampersands (&, &&) show the corresponding differences for the
internalized binding. DTT, dithiothreitol.

Fig. 3. Effects of two alkylating agents on surface binding and inter-
nalization of
125
I-labeled hPYY (3–36) in gpY2-CHO cells. The labeling
at 50 p
M
of the Y2 agonist was carried out for 40 min at 37 °C,inthe
presence of the indicated concentrations of NEM, MTSET or DTT.
The separation of surface and internalized tracer was done as in Figs 1
and2.Inthesamesetofexperiments(n ¼ 3), the surface binding
of the Y2 agonist in the presence of 10 l
M
PAO was 101 ±
2.8 fmolÆmg
)1
cell protein. Significance in Tukey t-testsisindicatedin
Fig. 2. DTT, dithiothreitol.
2318 S. L. Parker et al. (Eur. J. Biochem. 269) Ó FEBS 2002
saturated within 20 min at 24 °C, and in less than 10 min at
37 °C (Fig. 5A). More than 50% of the total increase in
surface gpY2-CHO sites by PAO was observed within
3 min of labeling at any temperature (Fig. 5A). A similar
fast activation was observed for Y2 receptors of rat
forebrain cells (not shown). This was contrasted by a
gradual, steady accumulation of surface Y1 sites in gpY1-
CHO cells at 3 l
M
filipin or 10 l
M
PAO, related to

inhibition of Y1 receptor internalization by these agents
(Fig. 5B). With the gpY1-CHO receptor (which is inten-
sively internalized and recycled, as different from the gpY2
receptor expressed in the same cell type [12]), there was a
gradual relative increase (over eightfold) in labeled surface
sites between 3 and 60 min of incubation at 24 °C in the
presence of either filipin III or PAO (Fig. 5B).
DISCUSSION
Several rhodopsin-family receptors were described as partly
cryptic, hidden, masked, or compartmentalized, for example
the pituitary LHRH [25], the protease-activated sevenfold
receptors such as the thrombin receptor [26], the a2-
adrenergic receptor reserve (60–70% of all sites [27]), and
the 5-HT1B receptor reserve (up to 90% of the total
receptor [28]). The muscarinic acetylcholine receptor, the
classic example of an aggregated rhodopsin-like receptor, is
usually examined in relation to its sequestration by agonists.
However, its massive aggregation by agrins, which also
involves cell-surface dystroglucans [29], could induce a
degree of constitutive, agonist-unrelated sequestration.
Among the Y receptors, the hypothalamic Y2 receptors
induced by estrogen show fast density changes by pro-
gesterone [30], possibly related to receptor masking. In this
study, competition by Y2 agonist after treatment by PAO
indicated an essentially normal affinity for the unmasked Y2
sites, which should derive from a constitutively sequestered
membrane compartment.
This work finds a considerable activation of cell mem-
brane Y2 sites by a vicinal dithiol-bridging agent, and a lower
activation by the alkylating agent NEM, or by the choles-

terol-complexing agent filipin III. With PAO, the cell-surface
activation reaches  50% of the receptor numbers found in
particulates derived by mechanical disruption of the cells. In
cell cultures, many of the surface receptors may not be
readily available for interaction with larger peptidic ligands
due to cell–cell interactions or interactions with the substra-
tum. It is therefore reasonable to assume that the arsenical
would expose a majority of the sites that can be labeled in the
absence of cell disruption by 34–36-residue peptides used for
the Y2 labeling. Lack of Y2 activation by the restricted
membrane-access alkylator MTSET [24] indicates a large
role for a cell membrane compartment in the masking of Y2
sites. From our previous work [14], a substantial part of Y2
receptors in this compartment should be aggregated and
anchored to cytoskeletal proteins, some of which contain
PAO-sensitive vicinal dithiol and even trithiol motifs.
Activation of masked populations of surface receptor
sites by PAO was shown previously for macroglobulin,
Fig. 4. Unmasking of surface Y2 sites in gpY2-CHO cells by filipin III
and effect of cholesterol. The cell monolayers were labeled by
125
I-labeled hNPY at 50 p
M
for 40 min at 37 °C at 1, 3, 10 or 30 l
M
filipin III (FIII) without or with 10 l
M
cholesteryl hemisuccinate
(Chol), and the tracer attached to surface receptors was extracted by
acid saline at 0–4 °C (see Materials and methods). Unmasking by

10 l
M
PAO resulted in surface binding of 90 ± 1.47 fmolÆmg
)1
pro-
tein without Chol, and of 89.3 ± 0.9 fmolÆmg
)1
protein with 10 l
M
Chol (n ¼ 3). Significance vs. the control binding was evaluated as in
Fig. 1.
Fig. 5. Comparative dynamics of labeling of Y2 sites in gpY2-CHO cells
and of Y1 sites in gpY1-CHO cells in the presence of phenylarsine oxide
or filipin III. The ligands (see below) were used at 50 p
M
for the indi-
cated periods of time. After removal of the unbound tracer and
washing, the cells were extracted with cold acid saline to separate the
cell surface-bound and internalized ligand (see Materials and meth-
ods). The results are expressed as percent of control labeling. The data
are averages of three experiments. (A) Surface labeling (ext) and
internalization (int) of
125
I-labeled hPYY(3–36) in gpY2-CHO cells at
10 l
M
PAO. The control labeling at 60 min was 20.3 ± 0.6 and
6.9 ± 0.23 (37 °C), and 24 ± 0.83 and 3.3 ± 0.15 fmolÆmg
)1
total

cell protein (24 °C) for the cell-surface and internalized fraction,
respectively. For any time point, the elevation of Y2 binding in the
presence of PAO was highly significant vs. the respective control
binding in Tukey t-testing. (B) Surface labeling (ext) and internaliza-
tion (int) of
125
I-labeled hNPY in gpY1-CHO cells at 24 °C in the
presence of 10 l
M
PAO or 3 l
M
filipin III. After 60 min at 24 °C,the
control labeling was 7.5 ± 0.23 and 37.7 ± 0.3 fmolÆmg
)1
total cell
protein for the cell-surface and internalized fraction, respectively. Note
that the internalized fraction of the Y1 binding with either inhibitor
was less than 5% of control values.
Ó FEBS 2002 Masked Y2 NPY receptors (Eur. J. Biochem. 269) 2319
transferrin and mannose-tipped glycoprotein receptors [31],
some of which are C-lectins possessing internal vicinal
dithiols [32], and could be activated by shedding or
disengaging the membrane neighbors through bridging by
PAO. This study finds that the surface Y2 binding to either
CHO cells or rat forebrain cells is strongly activated by PAO
below 100 l
M
. The Y2 receptor that contains no vicinal
dithiols [17] is inhibited by PAO only above 1 m
M

, i.e. at
molarities many orders of magnitude in excess of those
producing the maximum unmasking of cell-surface Y2 sites.
The activation of the Y2 receptor by PAO could mainly
result from alterations in arrangement of neighbors posses-
sing vicinal dithiols.
The communication between the extracellular matrix
(ECM) and the actin cytoskeleton (especially the a-actinin
component) indicates a direct involvement of basal lamina
molecules such as fibronectin, collagen IV, or laminin in
cytoskeletal handling of the plasma membrane nicotinic
receptor [33]. This could also apply to the Y2 receptor. The
shear-stress signal transduction proposed by Kano et al.
[34] could relate to the known aggregation of Y2 receptors
[14], and to an involvement of basement membrane
constituents. The shear can result from PAO-imposed
bridging of vicinal cysteines in ECM molecules, as well as of
tandem dithiols in a-actinins, adaptins and selectins. A role
for single intramembrane thiols could also be assumed,
based on the lack of Y2 activation by MTSET, and limited
activation by NEM, an alkylator that would access both the
exposed and the membrane-hidden thiols, but in a different
time frame [23]. Examination of the activity of the alkylating
agents, however, is complicated by their partial inactivation
of Y2 binding at molarities below 1 m
M
([14]; this work).
A thiol/disulfide redox system could regulate cell surface
Y2 availability mainly through neighboring or interacting
membrane proteins.

De-anchoring by PAO should also be considered in the
context of thiol-disulfide redox equilibria. Molecules sensi-
tive to trivalent arsenicals could be sought especially among
the strongly expressed adhesion and interaction factors such
as integrins (e.g. a1-integrin, a laminin and collagen
receptor), cadherins, and glypicans. Adhesion system
ligands possessing multiple vicinal dithiols such as melusin
[35], and multifunctional receptor/ligand proteins, e.g.
laminins, could also be modified by PAO [36], and this
might change the membrane protein arrangement in the
vicinity of Y2 sites. Anchoring of selectins (e.g.
L
-type; [37]),
as well as of other adhesion molecules [38] can be reduced by
dithiol bridging, and this type of change might alter the
availability of Y2 sites for agonist binding.
However, PAO can also act on intramembrane and
intracellular sites, as it penetrates the cell membrane with
ease, and does accumulate in the cytosol [39]. Among cell
membrane molecules that can be affected by the arsenical to
alter the state of aggregation of the Y2 receptor, certain
types of phosphatases could be of importance [40]. This,
however, may not be connected directly to the Y2 receptor,
which is poorly internalized in response to agonist binding
([12]; this work). Most of the activation of the Y2 sites
appears to be related to unmasking of occluded surface
receptors, and is also accomplished by shearing involved in
cell homogenization. Alkylators and PAO might also
increase cell permeability, as observed in the case of
occludin proteolysis [41]. Dethiolation of protein disulfides

via thioreductase could also be sensitive to PAO [42], and
the loss of disulfide dynamics could be partly responsible for
the observed Y2 accumulation. Stretch receptors and
mechanoreceptors could also participate in regulation of
Y2 sites. Tight junction-permeability could be increased by
tyrosine phosphorylation [43], to loosen the structure of
these aggregates, and PAO might act to improve the
phosphorylation by inhibiting tyrosine protein phospha-
tases [44].
Decrease in affinity of cell-surface Y2 sites after PAO is
small, and could mainly reflect disaggregation of the sites.
The change should be related to modifications of the
membrane environment of the receptor, as there are no
vicinal cysteines in the molecule of the rodent Y2 [17].
Unlike the metabotropic glutamate receptors or the adren-
ergic receptors, the Y receptors, except the Y5 [45], do not
contain vicinal cysteines that could impart a particular
sensitivity to PAO in ligand binding. Thus, the particulate
Y2 has a PAO K
I
above 1 m
M
, the Y1 of  400 l
M
, while
the Y5 shows a K
I
of only about 10 l
M
[46] (implicating the

seventh transmembrane segment in Y5 ligand binding).
Various proteasome subunits possess vicinal cysteines
[47], which can be modified by PAO to alter either the
proteolytic function, the anchoring, or the assembly of
proteasome complexes. The fast activation of Y2 binding by
PAO would indicate cell membrane rather than intracellular
targets. However, proteasomes are known to be associated
with cell membrane as well as with intracellular membrane
systems. Intramembrane targets of PAO could also include
G-protein b-andc-subunits that contain vicinal cysteines,
as well as some rab and ras G-proteins.
The stimulation of Y2 binding by filipin should not result
from accumulation of surface sites (as we have shown in
parallel experiments for the rapidly internalizing Y1 recep-
tor expressed in CHO cells), but rather from a direct
unmasking due to complexing of membrane cholesterol, as
cholesterol was able to abolish the effect of the polyene. This
may involve glycosylphosphatidylinositol anchors, known
to undergo a constitutive cholesterol-dependent sequestra-
tion into early endosomes [48].
The Y2 receptor activation by alteration of sulfhydryl or
cholesterol balance was found in this study for cells with
quite different plasma membrane systems. The CHO cells
are of epithelial derivation. The forebrain cells expressing
the Y2 receptor might, in addition to neuronal cells [49], also
include glia, in an analogy to kidney epithelia [50]. The Y2
activation indicates involvement of a general mechanism
that can operate across cell types to ensure rapid engage-
ment of a sequestered receptor pool in response to stimuli
that might range from micromechanical to oxidoreductive.

This segregation is not present with rapidly recycling Y1
receptors, but could be shared by other receptors that show
low rates of ligand-induced internalization [12].
ACKNOWLEDGEMENTS
This research was partly supported by NIH grants 13703 and 12844,
and by State of Tennessee Educational Funds.
REFERENCES
1. Mukherjee, S., Ghosh, R.N. & Maxfield, F.R. (1997) Endocytosis.
Physiol. Rev. 77, 759–803.
2320 S. L. Parker et al. (Eur. J. Biochem. 269) Ó FEBS 2002
2. Ferguson, S.S. (2001) Evolving concepts in G protein-coupled
receptor endocytosis: the role in receptor desensitization and
signaling. Pharmacol. Rev. 53, 1–24.
3. Wei, H.B., Yamamura, H.I. & Roeske, W.R. (1994) Down-
regulation and desensitization of the muscarinic M1 and M2
receptors in transfected fibroblast B82 cells. Eur. J. Pharmacol.
268, 381–391.
4. Tsuga, H., Kameyama, K., Haga, T., Honma, T., Lameh, J. &
Sadee, W. (1998) Internalization and down-regulation of human
muscarinic acetylcholine receptor m2 subtypes. Role of third
intracellular m2 loop and G protein- coupled receptor kinase 2.
J. Biol. Chem. 273, 5323–5330.
5. Parker, S.L., Parker, M.S., Kane, J.K. & Li, M.D. (2001) Inter-
nalization of neuropeptide Y (NPY) Y1 and Y2 receptors in dis-
persed rat forebrain cells. Society for Neuroscience 2001 Annual
Meeting, San Diego, CA, Abstract 461.7.
6. Oksche,A.,Boese,G.,Horstmeyer,A.,Furkert,J.,Beyermann,
M.,Bienert,M.&Rosenthal,W.(2000)Lateendosomal/lyso-
somal targeting and lack of recycling of the ligand- occupied
endothelin B receptor. Mol. Pharmacol. 57, 1104–1113.

7. Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J.,
Bloom, S.R. & Polak, J.M. (1983) Neuropeptide Y distribution in
the rat brain. Science 221, 877–879.
8. Kalra, S.P., Dube, M.G., Pu, S., Xu, B., Horvath, T.L. & Kalra,
P.S. (1999) Interacting appetite-regulating pathways in the
hypothalamic regulation of body weight. Endocrinol. Rev. 20,
68–100.
9. Parker, R.M. & Herzog, H. (1999) Regional distribution of
Y-receptor subtype mRNAs in rat brain. Eur. J. Neurosci. 11,
1431–1448.
10. Aicher, S.A., Springston, M., Berger, S.B., Reis, D.J. & Wahles-
tedt, C. (1991) Receptor-selective analogs demonstrate NPY/PYY
receptor heterogeneity in rat brain. Neurosci. Lett. 130, 32–36.
11. Parker, E.M., Balasubramaniam, A., Guzzi, M., Mullins, D.E.,
Salisbury, B.G., Sheriff, S., Witten, M.B. & Hwa, J.J. (2000)
[D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide
Y Y(5) receptor against with dramatic effects on food intake.
Peptides 21, 393–399.
12. Parker, S.L., Kane, J.K., Parker, M.S., Berglund, M.M., Lundell,
I.A. & Li, M.D. (2001) Cloned neuropeptide Y (NPY) Y1 and
pancreatic polypeptide Y4 receptors expressed in Chinese hamster
ovary cells show considerable agonist-driven internalization, in
contrast to the NPY Y2 receptor. Eur. J. Biochem. 268, 877–886.
13. Allen, J.M., Ferrier, I.N., Roberts, G.W., Cross, A.J., Adrian,
T.E., Crow, T.J. & Bloom, S.R. (1984) Elevation of neuropeptide
Y (NPY) in substantia innominata in Alzheimer’s type dementia.
J. Neurol. Sci. 64, 325–331.
14. Parker, S.L. & Parker, M.S. (2000) Ligand association with the
rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y
(NPY) receptors shows large subtype-related differences in sensi-

tivity to chaotropic and alkylating agents. Regul. Pept. 87, 59–72.
15. Parker, S.L., Parker, M.S., Sweatman, T. & Crowley, W.R. (1998)
Characterization of G-protein and phospholipase C-coupled
agonist binding to the Y1 neuropeptide Y receptor in rat brain.
Sensitivity to G-protein activators and inhibitors and to inhibitors
of phospholipase C. J. Pharmacol. Exptl Therap. 286, 1–12.
16. Berglund, M.M., Holmberg, S.K., Eriksson, H., Gedda, K.,
Maffrand, J.P., Serradeil-Le Gal, C., Chhajlani, V., Grundemar,
L. & Larhammar, D. (1999) The cloned guinea pig neuropeptide
Y receptor Y1 conforms to other mammalian Y1 receptors.
Peptides 20, 1043–1053.
17. Sharma, P., Holmberg, S.K., Eriksson, H., Beck-Sickinger, A.G.,
Grundemar, L. & Larhammar, D. (1998) Cloning and functional
expression of the guinea pig neuropeptide Y Y2 receptor. Regul.
Pept. 75–76, 23–28.
18. Haigler, H.T., Maxfield, F.R., Willingham, M.C. & Pastan, I.
(1980) Dansylcadaverine inhibits internalization of 125I-
epidermal growth factor in BALB 3T3 cells. J. Biol. Chem. 255,
1239–1241.
19. Beck-Sickinger, A.G., Wieland, H.A., Wittneben, H., Willim,
K.D.,Rudolf,K.&Jung,G.(1994)Complete
L
-alanine scan of
neuropeptide Y reveals ligands binding to Y1 and Y2 receptors
with distinguished conformations. Eur. J. Biochem. 225, 947–958.
20. Parker, M.S., Berglund, M.M., Lundell, I. & Parker, S.L. (2001)
Blockade of pancreatic polypeptide-sensitive neuropeptide Y
(NPY) receptors by agonist peptides is prevented by modulators of
sodium transport. Implications for receptor signaling and regu-
lation. Peptides 22, 887–898.

21. Munson, P.J. & Rodbard, D. (1980) LIGAND: a versatile com-
puterized approach for characterization of ligand-binding pro-
teins. Anal. Biochem. 107, 220–239.
22. Zar, J.H. (1984) Biostatistical Analysis. Prentice Hall, Englewood
Cliffs, NJ.
23. Zarbiv, R., Grunewald, M., Kavanaugh, M.P. & Kanner, B.I.
(1998) Cysteine scanning of the surroundings of an alkali-ion
binding site of the glutamate transporter GLT-1 reveals a con-
formationally sensitive residue. J. Biol. Chem. 273, 14231–14237.
24. Karlin, A. & Akabas, M.H. (1998) Substituted-cysteine accessi-
bility method. Methods Enzymol. 293, 123–145.
25. Leblanc, P., L’Heritier, A., Rasolonjanahary, R. & Kordon, C.
(1994) Neuropeptide Y enhances LHRH binding to rat gonado-
trophs in primary culture. Neuropeptides 26, 87–92.
26. Kawabata, A. & Kuroda, R. (2000) Protease-activated receptor
(PAR), a novel family of G protein-coupled seven trans-mem-
brane domain receptors: activation mechanisms and physiological
roles. Jpn J. Pharmacol. 82, 171–174.
27. Adler, C.H., Meller, E. & Goldstein, M. (1987) Receptor reserve at
the alpha-2 adrenergic receptor in the rat cerebral cortex.
J. Pharmacol. Exp. Ther. 240, 508–515.
28. Adham, N., Ellerbrock, B., Hartig, P., Weinshank, R.L. &
Branchek, T. (1993) Receptor reserve masks partial agonist
activity of drugs in a cloned rat 5-hydroxytryptamine1B receptor
expression system. Mol. Pharmacol. 43, 427–433.
29. Montanaro, F., Gee, S.H., Jacobson, C., Lindenbaum, M.H.,
Froehner, S.C. & Carbonetto, S. (1998) Laminin and alpha
dystroglycan mediate acetylcholine receptor aggregation via a
MuSK-independent pathway. J. Neurosci. 18, 1250–1260.
30. Parker, S.L., Carroll, B.L., Kalra, S.P., St-Pierre, S., Fournier, A.

& Crowley, W.R. (1996) Neuropeptide Y Y2 receptors in
hypothalamic neuroendocrine areas are up-regulated by estradiol
and decreased by progesterone cotreatment in the ovariectomized
rat. Endocrinology 137, 2896–2900.
31. Kaplan, J., Ward, D.M. & Wiley, H.S. (1985) Phenylarsine oxide-
induced increase in alveolar macrophage surface receptors: evi-
dence for fusion of internal receptor pools with the cell surface.
J. Cell Biol. 101, 121–129.
32. McKay, P.F., Imami, N., Johns, M., Taylor-Fishwick, D.A.,
Sedibane, L.M., Totty, N.F., Hsuan, J.J., Palmer, D.B., George,
A.J., Foxwell, B.M. & Ritter, M.A. (1998) The gp200-MR6
molecule which is functionally associated with the IL-4 receptor
modulates B cell phenotype and is a novel member of the human
macrophage mannose receptor family. Eur. J. Immunol. 28, 4071–
4083.
33. Dmytrenko, G.M. & Bloch, R.J. (1993) Evidence for transmem-
brane anchoring of extracellular matrix at acetylcholine receptor
clusters. Exp. Cell Res. 206, 323–334.
34. Kano, Y., Katoh, K. & Fujiwara, K. (2000) Lateral zone of cell-
cell adhesion as the major fluid shear stress-related signal trans-
duction site. Circ. Res. 86, 425–433.
35. Brancaccio,M.,Guazzone,S.,Menini,N.,Sibona,E.,Hirsch,E.,
De Andrea, M., Rocchi, M., Altruda, F., Tarone, G. & Silengo, L.
(1999) Melusin is a new muscle-specific interactor for beta (1)
integrin cytoplasmic domain. J. Biol. Chem. 274, 29282–29288.
Ó FEBS 2002 Masked Y2 NPY receptors (Eur. J. Biochem. 269) 2321
36. Bernier,S.M.,Utani,A.,Sugiyama,S.,Doi,T.,Polistina,C.&
Yamada, Y. (1995) Cloning and expression of laminin alpha 2
chain (M-chain) in the mouse. Matrix Biol. 14, 447–455.
37. Bennett, T.A., Edwards, B.S., Sklar, L.A. & Rogelj, S. (2000)

Sulfhydryl regulation of
L
-selectin shedding: phenylarsine oxide
promotes activation-independent
L
-selectin shedding from leuko-
cytes. J. Immunol. 164, 4120–4129.
38. Arribas, J., Coodly, L., Vollmer, P., Kishimoto, T.K., Rose-John,
S. & Massague, J. (1996) Diverse cell surface protein ectodomains
are shed by a system sensitive to metalloprotease inhibitors.
J. Biol. Chem. 271, 11376–11382.
39. Frost, S.C. & Schwalbe, M.S. (1990) Uptake and binding of
radiolabelled phenylarsine oxide in 3T3-L1 adipocytes. Biochem.
J. 269, 589–595.
40. Dai, Z. & Peng, H.B. (1998) A role of tyrosine phosphatase in
acetylcholine receptor cluster dispersal and formation. J. Cell Biol.
141, 1613–1624.
41. Wachtel, M., Frei, K., Ehler, E., Fontana, A., Winterhalter, K. &
Gloor, S.M. (1999) Occludin proteolysis and increased perme-
ability in endothelial cells through tyrosine phosphatase inhibition.
J. Cell Sci. 112, 4347–4356.
42. Park, E.M. & Thomas, J.A. (1989) The mechanisms of reduction
of protein mixed disulfides (dethiolation) in cardiac tissue. Arch.
Biochem. Biophys. 274, 47–54.
43. Staddon, J.M., Herrenknecht, K., Smales, C. & Rubin, L.L.
(1995) Evidence that tyrosine phosphorylation may increase tight
junction permeability. J. Cell Sci. 108, 609–619.
44. Durden, D.L., Rosen, H., Michel, B.R. & Cooper, J.A. (1994)
Protein tyrosine phosphatase inhibitors block myeloid signal
transduction through the Fc gamma RI receptor. Exp. Cell Res.

211, 150–162.
45. Herzog, H., Darby, K., Ball, H., Hort, Y., Beck-Sickinger, A. &
Shine, J. (1997) Overlapping gene structure of the human neuro-
peptide Y receptor subtypes Y1 and Y5 suggests coordinate
transcriptional regulation. Genomics 41, 315–319.
46. Parker,E.M.,Lundell,I.,Berglund,M.M.&Parker,S.L.(2001)
Internalization of pancreatic polypeptide Y4 receptors: correlation
of receptor intake and affinity. Society for Neuroscience 2001
Annual Meeting, San Diego, CA, Abstract 461.8.
47. Foss, G.S., Larsen, F., Solheim, J. & Prydz, H. (1998) Constitutive
and interferon-gamma-induced expression of the human protea-
some subunit multicatalytic endopeptidase complex-like 1. Bio-
chim. Biophys. Acta 1402, 17–28.
48. Mayor, S., Sabharanjak, S. & Maxfield, F.R. (1998) Cholesterol-
dependent retention of GPI-anchored proteins in endosomes.
EMBO J. 17, 4626–4638.
49. Bleakman, D., Colmers, W.F., Fournier, A. & Miller, R.J. (1991)
Neuropeptide Y inhibits Ca
2+
influx into cultured dorsal root
ganglion neurones of the rat via a Y2 receptor. Br.J.Pharmacol.
103, 1781–1789.
50. Sheikh, S.P., Sheikh, M.I. & Schwartz, T.W. (1989) Y2-type
receptors for peptide YY on renal proximal tubular cells in the
rabbit. Am. J. Physiol. 257, F978–F984.
2322 S. L. Parker et al. (Eur. J. Biochem. 269) Ó FEBS 2002