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-

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

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

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

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

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

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

) 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

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

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

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

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

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

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

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.

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-

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.

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.