MINIREVIEW SERIES

Orphan GPCRs in the regulation of sleep and circadianrhythmOlivier Civelli

Department of Pharmacology and Department of Developmental and Cell Biology, University of California, Irvine, CA, USA

The hypothalamus is the brain center that is most

important in regulating sleep duration and circadian

rhythm. Some 75 years ago, it was predicted that the

rostral hypothalamus contains the sleep-promoting

neurons, while the posterior hypothalamus contains

wakefulness-promoting neurons [1]. Sleep is controlled

by two processes, a homeostatic and a circadian timing

process, which together determine the propensity,

length, and incidence of episodes and intensity of sleep

[2]. Sleep itself is divided into two major phases, non-

rapid eye movement (NREM) and rapid eye movement

(REM) sleep [3]. Modulation of these phases and of

the circadian rhythms relies on several transmitters

which together generate and maintain sleep [4].

The sleep-producing neurons are c-aminobuty-

rate(GABA)-ergic cells [3,4]. They induce sleep by

inhibiting cells that are involved in arousal functions.

The wakefulness-promoting neurons on the other hand,

rely on several transmitters (Fig. 1). The peduncolo-

pontine and laterodorsal tegmental (PPT–LDT) nuclei

rely on acetylcholine and fire rapidly during wakeful-

ness and REM sleep but become inactive during

NREM sleep. It projects to the thalamus, in particular

to the reticular nucleus, which is thought to be critical

in activating thalamocortical transmission. Three other

distinct nuclei exhibit similar activity during the wake-

sleep phases. The tuberomammilary nucleus (TMN) is

a histaminergic nucleus and plays a major role in the

maintenance of wakefulness. Inhibition of their activity

by GABAergic cells appears to be closely linked to slee-

piness, as evidenced by the drowsiness elicited by anti-

histamine drugs. The locus coeruleus (LC), center of

norepinephrine production, is active during wakeful-

ness, displays low activity during NREM sleep and is

inactive during REM sleep. The raphe nucleus, which

relies on serotonin as transmitter, is also inactive dur-

ing sleep, in particular during REM sleep. Its inactivity

allows electrical activity to propagate from the pons to

the thalamus and cortex inducing eye movement and

twitches. Neurons from the TMN, LC and dorsal raphe

fire fastest during wakefulness, slow down during

NREM and nearly stop firing during REM sleep.

While most sleep research has focused on the actions

of neurotransmitters, an increasing amount of data

point at neuropeptides as being important in modula-

ting the sleep-wake cycle. Neuropeptides exert their

actions by activating G-protein coupled receptors

(GPCRs). Interestingly, most of these sleep-regulating

neuropeptides were discovered recently, not for their

activities at regulating sleep, but as the natural ligands

of orphan GPCRs.

The human genome expresses some 800 GPCRs of

which some 360 are activated by transmitters. Ten years

ago, half of these GPCRs had not been matched to any

known transmitters; they became to be known as

orphan GPCRs [5]. Because of their intrinsic receptor

nature, a strategy was developed to use the orphan

GPCRs as targets in the discovery of novel transmitters.

In short, orphan GPCRs are heterologously expressed

in cells in culture and subjected to tissue extracts expec-

ted to contain their natural transmitters. Activation of

the orphan GPCRs are monitored through their second

messenger responses. This strategy, first reported in

1995, has led to the discovery of 10 novel neuropeptides.

It has also allowed matching of several orphan GPCRs

to neuropeptides that had been described previously [5].

Novel neuropeptides discovered as ligands of orphan

GPCRs have unknown functions. Finding these requires

experimental studies that are predominantly directed by

the anatomical localization analyses of the sites of syn-

thesis and the sites of action of the novel neuropeptides

in the central nervous system (CNS). But ultimately, it

is the administration of the novel neuropeptide to ani-

mals and ⁄or the engineering of knockout mouse strains

that reveals the function of the novel neuropeptide sys-

tem. Surprisingly a series of novel neuropeptide systems

were found to be implicated in sleep and wakefulness.

The first deorphanized GPCR system to be shown to

modulate sleep was the hypocretin ⁄orexin (Hcrt ⁄orx)system. This system relies on the action of two closely

related neuropeptides at two sequentially similar

GPCRs. This system originates in the lateral hypothala-

mus and projects to throughout the whole brain but in

particular to the LC, dorsal raphe and PPT–LDT. That

doi:10.1111/j.1742-4658.2005.04867.x

FEBS Journal 272 (2005) 5673–5674 ª 2005 FEBS 5673

the Hcrt ⁄orx system is a major modulator of sleep has

been demonstrated by the discovery that animals, in

which one of the Hcrt receptors is inactive (Hcrt2), are

narcoleptic and that most human narcoleptic patients

have no detectable circulating Hcrt and exhibit a

reduced number of Hcrt neurons. This system is

reviewed in this series by de Lecea & Sutcliffe.

Another novel neuropeptide system that modulates

sleep is the neuropeptide S (NPS) system. NPS is syn-

thesized in several parts of the CNS but in particular

in a nucleus anatomically associated with the LC. Acti-

vation of the NPS system promotes wakefullness by

decreasing the NREM and REM stages of sleep. This

system is reviewed in this series by Reinscheid & Xu.

The urotensin II (UII) receptor, a GPCR deorphanized

in 1999, has been shown to be selectively expressed in the

cholinergic LDT–PPT neurons in the CNS. These neu-

rons fire during REM sleep. The UII receptor acts as a

presynaptic receptor in the LDT–PPT and consequently

activation of this system has been shown to increase REM

sleep. This system is reviewed by Nothacker & Clark.

However, there are other orphan GPCR systems that

impact sleep. The prolactin-releasing peptide (PrRP)

system is one of these. In the CNS, the PrRP receptor is

predominantly expressed in the reticular nucleus of the

thalamus. This is the thalamic relay nucleus of the choli-

nergic LDT–PPT and it has been shown that activation

of this system induces sleep [6].

Sleep regulation is directly linked to the circadian

rhythm. The circadian rhythm is orchestrated in the

suprachiasmatic nucleus (SCN) which provides the

genetically based clock. The SCN clock relies on posit-

ive and negative feedback loops involving the time-

dependent transcription of a series of genes [7]. The

factors responsible for the output of the SCN clock

however, were unknown until recently when prokineti-

cin 2 (PK2) was shown to be such a factor. PK2 was

discovered as the natural ligand of an orphan GPCR

and is reviewed in this series by Zhou.

This minireview series is intended to present the

impact that novel neuropeptides have on our under-

standing of the sleep-wake cycle. This is not the only

field on which novel neuropeptides have had an

impact. Most notably, our understanding of the regu-

lation of feeding has greatly gained from the discover-

ies of novel neuropeptides. These reviews therefore

serve as examples of the importance of the emerging

field of the natural ligands of orphan GPCRs.

References

1 Von Economo C (1930) Sleep as a problem of localiza-

tion. J Nerv Ment Dis 71, 249–259.

2 Borbely AA (1982) A two process model of sleep regula-

tion. Hum Neurobiol 1, 195–204.

3 Saper CB, Chou TC & Scammell TE (2001) The sleep

switch: hypothalamic control of sleep and wakefulness.

Trends Neurosci 24, 726–731.

4 Siegel JM (2004) The neurotransmitters of sleep. J Clin

Psych 65, 4–7.

5 Civelli O, Nothacker HP, Saito Y, Wang Z, Lin S &

Reinscheid RK (2001) Discovery of novel neurotransmit-

ters as natural ligands of orphan G protein-coupled

receptors. Trends Neurosci 24, 230–237.

6 Lin SHS, Arai A, Espana RA, Berridge CW, Leslie F,

Huguenard J, Vergnes M & Civelli O (2002) Prolactin-

releasing peptide (PrRP) promotes awakening and sup-

presses absence seizures. Neurosci 114, 229–238.

7 Reppert SM & Weaver DR (2002) Coordination of circa-

dian timing in mammals. Nature 418, 935–941.

Fig. 1. Sleep architecture and its transmitters. In green are the tra-

ditional transmitters: GABA, 4-aminobutyrate; Hist, histamine; NA,

norepinephrine; Ach, acetylcholine. The new tranmitters and their

sleep-related sites of synthesis: TMN, tuberomammilary nucleus;

Raphe, raphe nucleus; LC, locus coeruleus; PPT ⁄ LTD, peduncolo-

pontine and laterodorsal tegmental. The novel transmitters are in

red: HCRT, hypocretin ⁄ orexin; PK2, prokineticin 2; NPS, neuropep-

tide S; UII, urotensin II. - - - - - , sleep-producing pathways; ——,

wakefulness-producing pathway.

Olivier Civelli, PhD, is Professor of Pharmacology and the Eric L. and Lila D. Nelson Chair of Neuropharmacology at

University of California, Irvine. He is one of the pioneers who devised molecular strategies for understanding the

structure and function of drug receptors. He was first to clone a dopamine receptor, and deciphered their complexity.

He also devised and successfully applied the strategy that uses orphan GPCRs to discover novel neurotransmitters.

His present research aims at discovering new neuropeptides and at finding their biological significance. His discover-

ies continue to have important implications for the development of new therapeutic targets.

Sleep and circadian rhythm regulation by novel neuropeptides O. Civelli

5674 FEBS Journal 272 (2005) 5673–5674 ª 2005 FEBS