Tài liệu Báo cáo Y học: A pool of Y2 neuropeptide Y receptors activated by modifiers of membrane

PRIORITY PAPER

A pool of Y2 neuropeptide Y receptors activated by modifiers

of membrane sulfhydryl or cholesterol balance

Steven L. Parker1

, Michael S. Parker2

, Justin K. Kane1 and Magnus M. Berglund3

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: [email protected]

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)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02903.x