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Orphan seven transmembrane domain receptors: reversing

pharmacology* Ann Mills and Michael J. Duggan

The application of molecular genetic approaches to the study of seven

transmembrane domain receptors has allowed the cloning of many receptors for

which the ligand is initially unknown. These are commonly referred to as 'orphan

receptors', and several have subsequently proved to be important pharmacological

targets. This article discusses how these receptor sequences were isolated, and

presents some of the methods by which the corresponding ligands were identified.

These examples are used to propose a rational approach for the study of further

orphan receptors.

Originally, the cloning of seven transmembrane domain G protein-coupled receptors depended on the isolation and sequencing of the corresponding protein or the use of expression cloning techniques. However, when sequences for these receptors became available, it was apparent that there were significant sequence homologies between these receptors, particularly in their transmembrane domains. These homologies could be exploited to specifically clone related mem- bers of the superfamily by using techniques such as homology screening or polymerase chain reaction (PCR). Subsequently, these techniques have been modified to reduce their specificity (i.e. homology screening at low stringency or PC1K using degenerate primers) to allow isolation of sequences more distantly related to the known G protein-coupled receptors. Clones obtained in this way have been termed 'orphan receptors' because at the time of cloning their ligand is unknown, yet they have the potential to revol- utionize pharmacological research.

In their seminal paper on the use of PC1K to clone novel receptors, Libert et al. 1 cloned four orphan receptors from rat thyroid cDNA using oligo- nucleotide primers derived from regions of trans- membrane domains III and VI that are conserved between the muscarinic acetylcholine M1, neurokinin NK2, 5-hydroxytryptamine 5HTLa and [31- , [32- and c¢2-adrenoceptors. Three of these novel receptor

A. Mills is at the Glaxo Institute for Molecular Biology S.A., 14 Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland. M.J. Duggan is at The Speywood Laboratory, St George's Hospital Medical School, Cranmer Terrace, London, UK SW17 OQS.

*This is an updated version of an article that was first published in

Trends in Pharmacological &iences 14, 394-397 (1993).

sequences were subsequently identified as the 5 H T 1 D

and the adenosine A 1 and A 2 receptors 2-4, none of which had previously been cloned. Since this report, many other orphan receptors have been isolated (Table 1) and have been shown to be pharmacologi- cally important. However, a number of putative receptor clones remain to be characterized, and the major challenge in this line of research is how to identify the physiological ligand.

Circumstantial evidence For some orphan receptors, the clue that led to the

discovery of their ligand lay in their amino acid sequences, which had significant homology to for- merly identified receptors; notable examples are the 5HT1D receptor and the A 1 and A 3 receptors 5, all of which share 40-60% homology with known receptors of the same family. Interestingly, the cloned A 3 recep- tor does not appear to correspond to the putative A 3 receptor that had been predicted by pharmacological studies 6. Current methods for comparative sequence analysis cannot predict the ligand for a new receptor if the degree of homology is lower than 40-60%. However, with the development of new techniques such as hydrophobic cluster analysis 7, which combines homology comparisons with analysis of secondary structure, the predictive power of such comparisons may be improved. Although the extent of sequence homology between receptors may not be sufficient to assign an orphan receptor to a particular family, sequencing can reveal motifs associated with specific ligands. An aspartate residue in the third transmem- brane domain at position 113 of the [32-adrenoceptor is conserved in all adrenoceptors and in muscarinic, dopamine and 5-HT receptors, but does not generally appear in neuropeptide receptors 8.

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Table 1. Identified seven transmembrane domain orphan receptors a

Receptor Cloning strategy Host cell line Response Refs

Adrenocorticotropic hormone PCR Cloudman S91 SCAMP 12

Adenosine A 1 PCR COS-7 3

CHO-K1

A 2 PCR COS-7 4 Xenopus oocytes

A 3 PCR 5 COS-7 CHO-K1

Cannabinoid Homology CHO-K1

Melanocyte-stimulating hormone PCR HEK-293

Neuropeptide Y1 PCR COS-1

5-Hydroxytryptamine 5HT1A Homology COS-7

[3H]CGS21680 binding SCAMP

[3H]CGS21680 binding SCAMP

[3H]NECA binding [12Sl]APNEA binding SCAMP

SCAMP

SCAMP

[1251]peptide YY binding

[1251]iodocyanopindolol binding [3H]8-OH-DPAT binding

5HT1D PCR COS-7 [3H]LSD binding 2 Y1 "]'cAMP

9

12

10,11

14

Somatostatin SS 1 PCR CHO [1251]-[-i-yral]somatostatin 13 binding

aAbbreviations: NECA, 5'-N-ethylcarboxamidoadenosine; APNEA, N6-2-(4-aminophenyl)ethyladenosine; 8-OH-DPAT, 8-hydroxy-2-(di-n- propylamino)tetralin; LSD, lysergide; CHO, Chinese hamster ovary; PCR, polymerase chain reaction.

In the absence of significant sequence homology, mapping the tissue distribution of a novel receptor can be immensely helpful in receptor identification. The cannabinoid receptor was first cloned as an orphan receptor, SKI<6, from rat cerebral cortex cDNA by using low stringency homology screening with an oligonucleotide probe from the neurokinin NK 2 receptor 9. After testing a number of possible peptide ligands, the identity of the receptor was finally deduced from the presence of SKR6 m R N A both in cell lines and in regions of the brain that express cannabinoid binding sites. Localization was also the key to classification of the orphan receptor FC5 (Ref. 10) as a neuropeptide Y1 receptor; the expression pattern of FC5 mR_NA in rat brain, as determined by in situ hybridization, was recognized as resembling that of the Y1 receptor visualized by ligand auto- radiography 11. For many orphan receptors, the tissue localization is already partially known, since they have been cloned from a highly specialized tissue or organ. Thus, the melanocortin receptors were cloned from melanoma cDNA 12 and the somatostatin SS~ receptor from pancreatic islet cDNA 13.

Proving the case In all cases, the final proof of the identity of an

orphan receptor has come from expression of the

receptor in a cell line or in Xenopus laevis oocytes, fol- lowed by radioligand binding or functional assays with the appropriate ligand (Table 1). Importantly, these techniques can also be used for general screen- ing of potential ligands for their interaction with novel receptors. Screening for ligands by radioligand bind- ing assays is likely to be prohibitively expensive and, therefore, a functional approach is preferable. To date, only one orphan receptor has been identified by radio- ligand screening, in the absence of any other clues to its identity, and this was serendipitous. The human genomic clone G21 (P,.ef. 14) shared 45% homology with adrenoceptors and a moderate affinity for the [32-adrenoceptor antagonist radioligand [12sI]iodo- cyanopindolol. Fortuitously, this radioligand also has an affinity for 5-HT receptors, and orphan G21 was subsequently identified as a 5HTla receptor.

Several factors can comphcate the use oftransfected cell lines for ligand screening, including the presence of endogenous receptors for a variety of compounds and the level of expression of the heterologous recep- tor protein that can be attained. Many groups have used northern blotting to demonstrate the presence of orphan receptor m R N A and have then assumed that the protein is also present. Other studies, however, suggest that this is a dangerous assumption. The clonal pituitary cell line GH3 produces mR.NA for

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dopamine D 2 receptors but does not show any recep- tor binding is, while Chinese hamster ovary (CHO) cells transfected with D 4 receptors also produce D 4 m R N A but do not bind [3H]spiperone (A. Mills, unpublished). To overcome this problem, western blotting can be used to confirm expression of the novel receptor protein. If, as is likely, antibodies for the receptor itself are not available, an antigenic epitope can be attached to the receptor; an example is the eight amino acid FLAG sequence (DYKDDDA), which is recognized by commercially available antibodies (IBI, New Haven, USA). Addition of this sequence to the N-terminus of the f32-adrenoceptor allowed immuno- detection of the protein without changing the phar- macology of the receptor 16.

Ligand screening using a functional assay allows test- ing of known ligands as well as those not yet ident- ified, as 6ssue extracts can also be used. At present, it is impossible to predict the G protein-coupling of a receptor from its amino acid sequence, yet this approach relies on the ability of the novel receptor to couple to a signalling pathway in the chosen host cell line. Despite the wide variety of cell types that may be used, there are already receptors for which no coupling pathway has yet been found, namely the dopamine D 3 and somatostatin SS 1 receptors 17,18. How- ever, the majority of seven transmembrane domain receptors that have been expressed recombinantly have been shown to regulate adenylyl cyclase activity or to stimulate phosphoinositide metabolism.

A number of assays have recently been developed for measuring second messenger producuon in vitro. For example, McClintock et al. 19 have used an X. laevis melanocyte cell line to study the activity of recom- binant G protein-coupled receptors that modulate either cAMP or phosphoinositide production. In this system, the dispersion or aggregation of pigment-con- taining melanosomes is followed by measuring light transmission at a wavelength of 620 nm. Second mess- enger levels in mammalian cells can also be analysed using photometric methods. Himmler et al. 2° used the firefly luciferase gene under the control of several cAMP responsive elements to follow the stimulation of adenylyl cyclase by dopamine D 1 and D s receptors recombinantly expressed in C H O cells.

Summing up Although orphan receptor research is fraught with

technological pitfalls, it has a number of attractions. First, it has already allowed identification of receptor subtypes for known receptor classes that had not been suspected from pharmacological investigations[ Sec- ondly, it has permitted cloning of 'difficult' receptors where more conventional methods had failed. Finally, and most importantly, this technology may aid the discovery of receptors for new ligands, and these molecules may p~irticipate in as yet undiscovered intercellular pathways. The recent report of a brain constituent, anandamide, which binds to the canna- binoid receptor, has added fresh impetus to the search for natural ligands. Libert and colleagues 2I have

called the use of recombinant receptors to identify ligands 'reverse pharmacology', and this strategy looks set to uncover important new pharmacological targets.

Since this article was first published 22, other orphan receptors have been identified as pharmacologically important species. Notable amongst these are the K and 8 opioid receptors 23 and a novel cannabinoid receptor 24. This latter receptor is exclusively dis- tributed peripherally, and its discovery opens up the possibility of a new class of pharmaceutical agents. These advances serve to emphasize the importance of this line of research to the development of new therapies.

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1460-1468 3 Libert, F. et al. (1991) EMBOJ. 10, 1677-1682 4 Maenhaut, C. et al. (1990) Biophys. Biochem. Res. Commun. 173,

1169-1178 5 Zhou, Q-Y. etal. (1992) Proc. NatlAcad. Sd. USA 89, 7432-7436 6 Carruthers, A. M. and Fozard, J. tL. (1993) Trends PharmacoL Sci. 14,

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Pavirani, A. (1993)J. Recept. Res. 13, 179-194 8 Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J. and Sealfon,

S. C. (1992) D N A Cell Biol. 11, 1-20 9 Matsuda, L. A., Lolait, S.J., Brownstein, M. J., Young, A. C. and

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(1990) FEBS Lett. 271, 81-84 11 Larhammar, D. et al. (1992)J. Biol. Chem. 267, 10935-10938 12 Mountjoy, K. G., Robbins, L. S., Mortrud, M. T. and Cone, R. D.

(1992) Science 257, 1248-1251 13 Yamada, Y. et al. (1992) Proc. Natl Acad. Sci. USA 89, 251-255 14 Fargin, A. et al. (1988) Nature 335, 358-360 15 Missale, C. et al. (1991)J. Biol. Chem. 266, 23392-23398 16 Guan, X-M., Kobilka, T. S. and Kobilka, B. K. (1992)J. Biol. Chem.

267, 21995-21998 17 Sokoloff, P., Martres, M-P., Giros, B., Bouthenet, M-L. and

Schwartz, J. C. (1992) Biochem. Pharmacol. 43, 659-666 18 Rens-Domiano, S. et al. (1992) Mol. PharmacoL 42, 28-34 19 McChntock, T. S. et al. (1993) Anal. Biochem. 209, 298-305 20 Himmler, A., Stratowa, C. and Czernilofsky, A. P. (1993)J. Recept.

Res. 13, 79-94 21 Libert, F., Vassart, G. and Parmentier, M. (1991) Curt. Opin. CellBiol.

3, 218-223 22 Mills, A. and Duggan, M.J. (1993) Trends Pharmacol. Sci. 14, 394-397 23 Yasuda, K. et al. (1993) Proc. Natl Acad. Sci. USA 90, 6736-6740 24 Munro, S., Thomas, K. L. and Abu-Shaar, M. (1993) Nature 363,

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