J. OF RECEPTOR & SIGNAL TRANSDUCTION RESEARCH, 17(1-3), 545-550 (1997)

MINIREVIEW

ORPHAN RECEPTORS AND THEIR NATURAL LIGANDS

0. Civelli, H.P. Nothacker, A. Bourson, A. Ardati, F. Monsma and R. Reinscheid CNS Department

F. Hoffmann-La Roche Ltd CH-4070 Base1

INTRODUCTION

In the field of G protein-coupled receptors (GPCRs), the use of homology screening approaches has led to the identification of an increasing number of "orphan" receptors. These are proteins which exhibit the structural characteristics shared by all members of the supergene family (seven putative transmembrane domains, often sequence similarities with other GPCRs) but which, on the basis of their primary sequences, do not belong to any of the receptor subfamilies (i.e. biogenic amine, peptide families, etc). Because non-functional receptors would have diverged sequentially during evolution, we expect the orphan receptors to bind novel, thus far undescribed, primary messengers (transmitters, peptides or hormones). Of immediate consequence, we foresaw that the orphan receptors could serve as targets to identify their natural ligands. Therefore. we decided to use an orphan receptor as a molecular target to isolate a novel transmitter, i.e. its natural ligand.

IDENTIFICATION AND CHARACTERIZATION OF THE NATURAL LIGAND OF AN ORPHAN GPCR

LC132 (also called ORL-1 and others (1)) is a GPCR which exhibits about 65% of sequence identity with the three opioid receptors in its transmembrane domains. Yet it had been shown not to bind any of the natural enkephalin-

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Copyright C 1997 by Marcel Dekker. Inc

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containing peptides nor any of the synthetic opioid ligands. It was therefore an orphan receptor suited for our natural ligand search. Because of its similarities to the opioid receptors, we assumed that this receptor might also bind a peptidergic ligand and share a similar coupling mechanism to second messenger systems as in the case of the opioid receptors, i.e. inhibition of adenylyl cyclase activity. We therefore decided to monitor activation of this receptor through inhibition of forskolin-stimulated CAMP accumulation in cells expressing LC 132 heterologously. Because the LC132 M A is expressed in hypothalamus (2), we set out to purify its natural ligand(s) from hypothalami.

The LC132 receptor cDNA was transfected in CHO cells. A clonal cell line expressing significant levels of LC132 mRNA w2.s identified and used in all experiments in comparison with untransfected CHO cl:lls.

Porcine hypothalami were acetic acid-extracted and processed according to standard protocols for the isolation of neuropeptide:;. After adsorption over C18 silica gel and an ultrafiltration to remove materials bigger than 10 kDaltons, a cation-exchange chromatography step revealed, among others, two consecutive pools containing adenylyl cyclase inhibitory activity ir LC132-transfected but not in untransfected cells. The corresponding individual fractions were further fractionated through five steps of reversed-phase HF'LC and from these an active compound was finally purified to homogeneity. This material was analyzed by mass spectrometry and sequenced by Edman degradation and proved to be a peptide with the primary structure FGGFTGARKSAFWANQ. Final yields of this peptide were ca. 200 pmoles from 4.5 kg brain tissue (wet weight). Computer data bank searches revealed that this peptide had not been previously reported either as a unique entity or as part of a larger protein. This peptitle was named "orphanin FQ" (OFQ) to mark its orphaned origin and to outline the f rst and last amino acids of its sequence, it has also been referred to as nociceptin (3).

The striking features of OFQ are its similarities to the opioid peptides: i) its aminotenninal tetrapeptide FGGF similar to the canmical sequence YGGF of the opioid peptides; ii) two clusters of basic amino acid; which are present in the C- terminal part of OFQ are reminiscent of the arrangement of multiple positively charged residues in dynorphin A or P-endorphin; iii) the C-terminal portion OFQ

contains a repetitive sequence (ARKXARKXA) resembling the repeat found at the C-terminus of most mammalian P-endorphins (KNAXXKNA). These data suggest that OFQ may be sequentially (and thus evolutioriarily) related to the opioid

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ORPHAN RECEPTORS 547

peptides. However, we could not detect activity of OFQ at the opioid receptors ( I ) nor could we show binding of the classical opioid peptides to the LC132 receptor ( I ) , revealing a pharmacological divergence between this novel peptide and the opioid peptides.

To demonstrate that OFQ was the active component of the extract, a peptide of the same sequence was synthesized and shown to potently inhibit forskolin- stimulated cAMP accumulation in LC132 transfected CHO cells ( EC50 1.05 i 0.21 nM and a maximal effect of approximately 80% inhibition at 100 nM). This high potency is comparable to that of other neuropeptides for their receptors and indicates that this novel peptide is a natural ligand for the LC 132 receptor.

PHARMACOLOGICAL CHARACTERISTICS OF OF0

For a ligand to be specific to a receptor, it must bind this receptor with saturable, displaceable kinetics and with high affinity. To analyze the physical constants of the binding of OFQ to its receptor, we needed to develop a radioligand. Because the OFQ ligand does not contain Tyr residues, a series of peptide analogs were synthesized which are tyrosine-substituted at different positions. From these studies, the Y I4 substituted OFQ as well as the monoiodo- Y14 analog were shown to be agonists of almost equivalent potency in cAMP assays with EC50 values of 1.02 k 0.1 1 nmol/l and 1.32 k 0.79 nmol/l, respectively ( 1). The [ 1251]-labelled Y 14-substituted peptide displayed saturable and displaceable binding to membranes of LC 132 receptor transfected cells with a I Q of 0.1 k 0.02 nmol/l and a B,,, of 843 k 29.7 fmol/mg membrane protein ( I ) . These data confirm that OFQ binds the LC 132 receptor in a saturable manner and with high affinity, indicating that this novel peptide is a natural ligand to the opioid- like receptor. Furthermore, these experiments show that the Y 14-substituted peptide can be used as a radioligand to detect and quantify LC132 receptor levels.

PHYSIOLOGICAL PROFLE OF OF0

To be classified as a novel neurotransmitter, OFQ should regulate a physiological response(s). The in vivo activity of OFQ was evaluated in mice

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following intracerebroventricular (i.c.v.) and intrzthecal (i.t.) administration. Because of its sequence similarity to the opioid peptides, we decided to determine whether OFQ was involved in nociception and whether any specific behavioral effects could be observed. The peptide was administered at increasing doses (0.1- 10 nmol/2pl/mouse, i.c.v.) and the behavior of the animals was observed.

Locomotor activity was quantified in an automated activity monitor system. At the highest dose, the peptide induced a decrease in horizcntal and vertical activity (1). To examine possible analgesic effects, a hot-plate test was performed but no significant inhibition of nociception was found. Only at the highest dose (10 nmol/mouse, i.c.v.), did the animals appear to show an antinociceptive behavior which was most likely related to the decrease of loccmotor activity and muscular tone. We could therefore conclude that, in the hot-plate test, OFQ did not induce analgesia at doses that did not produce significant decrease in motor activity and muscular tone. On the other hand, when tested in tl-e tail-flick assay, OFQ was found to induce an hyperalgesic effect (0.3 - 10 nmoV5 pVmouse, i.c.v.). At a dose of 1 nmol the reaction time is reduced by about 7547 when compared to vehicle injected animals (1). These data therefore confirm that OFQ does not induce an analgesic response in viva. From these studies we can conclude that, despite its structural homology to the opioid peptides, OFQ appt:ars to be pharmacologically and physiologically distinct from the opioid peptides.

BIOSYNTHESIS OF OF0

Because, all bioactive peptides are synthesized in form of precursor polypeptides, we set out to clone the cDNA of precursor of OFQ (3,4). We found that OFQ is synthesized as part of a larger polypeptide (181 amino acids). The precursor exhibits all the characteristics expected of a neuropeptide precursor (signal sequence, pair of basic amino acid residues flziking the bioactive OFQ). In

this precursor the 28 residues located C-terminal to the OFQ sequence have been found to be totally conserved among different mammalian species and thus to be potentially part of another bioactive peptide. The preciirsor shares some interesting topological similarities to the opioid peptide precursors, in particular conservation of seven Cys residues at its N-terminus. These data further support a common evolutionary component between OFQ and the opioid peptides. The precursor

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ORPHAN RECEPTORS 549

cDNA was used to demonstrate that the novel peptide is synthesized in different tissues in the CNS ().

CONCLUSION: ORPHAN RECEPTORS AS TOOLS FOR THE IDENTIFICATION OF NOVEL NEUROTRANSMITTERS

Our results show that a natural ligand of the opioid-like orphan GPCR is the novel peptide OFQ. This peptide can be accepted as a novel neuropeptide since it specifically binds a receptor found in the CNS, since it is expressed in the CNS and since it has a distinct physiological effect..

This work represents an example of the use of orphan receptors to identify novel natural transmitters. In view of the numerous orphan receptors that have been and are continuing to be cloned, we can expect many novel transmitters to be discover via this approach in the near future. This will result in a surge of our knowledge on the diversity in intercellular signalling mechanisms and should lead to novel insights on complex or current poorly understood human disorders. Because many of these novel transmitters will affect brain function, some (or most probably their synthetic analogs) may also be misused.

The question of the nomenclature of these new transmitters will also be raised. We will most probably not find immediately a defined physiological function for the new transmitters. Therefore, the sole feature of these transmitters will be their structure and their origin, from an orphan receptor. They will all be “orphanins”. To differentiate the peptides among them we have chosen to specify the first one that we found by its first and last residues. In this respect, the discovery of these new transmitters will embellish the diversity of the GPCR nomenclature.

REFERENCES

1. Reinscheid, R.K.; Nothacker, H.-P.; Bourson, A.: Ardati, A.; Henningsen, R.A.; Bunzow, J.R.; Grandy, D.L.; Langen, H.; Monsma, F.J., Jr.; and Civelli, 0. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270,792-794, 1995.

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2 . Bunzow, J.R.; Saez, C.; Mortrud, M.; Bouvier, C.; Williams, J.T.; Low, M.; and Grandy, D.K. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a p, F or K opioid receptor type. FEBS Lett. 347, 284-288, 1994.

3 . Meunier, J.-C.; Mollerau, C.; Toll, L.; Suaudeau, C.; Moisand, C.; Alvinerie, P.; Butour, J.-L.; Guillemot, J.-C.; Ferrara, P.; Monsarrat, B.; Mazarguil, H.; Vassart, G.; Parmentier, M.; anc Costentin, J. Isolation and structure of the endogenous agonist of opioid re ceptor-like OlUl receptor. Nature 377, 532-535, 1995.

4 . Nothacker, H.P.; Reinscheid, R.K.; Mansour, A.; Henningsen, R.A.; Monsma, F.J.; Watson, S.J.; and Civelli, 0. Primary structure and tissue distribution of the orphanin FQ precursor. Submitted, 1996.

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