Urotensin II: fish neuropeptide catches orphan receptor

COMMENT and unnatural substitutes for each amino acid were identified based on how they fit into the model. Essential hydrogen donor and acceptor sites of the peptide backbone also had to be maintained11. The authors could identify approximately 30 suitable substitutions, which – when assembled combinatorially – can give rise to thousands of distinct compounds. The biological activity of a few selected compounds appears promising. One improvement compared with peptide antagonists is that the peptidomimetics inhibited T-cell responses at ratios as low as 1:1 to the antigenic peptide. Another even more important improvement is that the mimetics effectively inhibited T-cell responses to processed protein antigens (i.e. responses that mimic the in vivo situation). Such responses could not, or only marginally, be inhibited by peptide-based antagonists. According to the authors’ quantitation, the inhibitory potency of the presented mimetics is between several-100-fold and more than 1000-fold

higher than that of the most potent peptides. The specificity of inhibition was addressed; the antagonists exhibited different patterns of allele selectivity depending on the mimetic blocks used. This implies that selective antagonists could be ‘custom-made’ to any desired MHC allelic molecule. The antagonists block T-cell responses to virtually all antigens presented by the targeted MHC molecules, ranging from model protein antigens to type-II collagen, a candidate autoantigen in rheumatoid arthritis. Altogether, this work raises hopes that selective immunotherapy has moved a significant step forward from being an immunologist’s dream towards pharmacological reality. Selected references 1 Nepom, B.S. (1993) The role of the major histocompatibility complex in autoimmunity. Clin. Immunol. Immunopathol. 67, S50–S55 2 Falcioni, F. et al. (1999). Design of peptidomimetic compounds that inhibit antigen presentation by autoimmune diseaseassociated class II MHC molecules. Nat. Biotechnol. 17, 562–567

Urotensin II: fish neuropeptide catches orphan receptor Anthony P. Davenport and Janet J. Maguire

A.P. Davenport, Lecturer and Director, E-mail: apd10@ medschl.cam.ac.uk and J.J. Maguire, Senior Research Associate, BHF Human Cardiovascular Receptor Research Group, Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110, Addenbrooke’s Hospital, Cambridge, UK CB2 2QQ. E-mail: jjm1003@ medschl.cam.ac.uk

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Urotensin II (U-II), a cyclic dodecapeptide, was originally isolated from the urophysis, the hormone-secretory organ of the caudal neurosecretory system of teleost fish, and the sequence determined nearly 20 years ago1. Several structural forms of U-II have subsequently been reported in different species of fish and amphibians, with variation occurring in the first five to seven amino acids of the N-terminal; the C-terminal cyclic hexapeptide sequence is conserved across species (Fig. 1). In fish, the peptides have several actions, including general smooth muscle contracting activity, although responses vary between species and vascular beds1. Fish U-II (mainly from goby), has also been shown to possess constrictor activity in mammals, including major arteries from the rat, but the receptor mediating these peptide actions was unknown2–4. Recently, Ames and col-

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leagues5 identified a gene encoding a novel human, seven-transmembrane, G-protein-coupled receptor (GPCR) that has 75% sequence similarity to an orphan receptor GPR14 from the rat6 [which is identical to the rat SENR (sensory epithelium neuropetide-like receptor)]7. Ames et al.5 used cells expressing human GPR14 to screen over 700 potential agonists for the activating ligand and goby U-II was identified as a potent stimulator of Ca21 responses in these transfected cells5. This action was mimicked by the recently identified human U-II (Ref. 8), an 11 amino acid peptide that retains the cyclohexapeptide sequence of goby U-II. It is a testament to the intense competition and interest in identifying ligands for orphan receptors that within two months three other independent groups, Nothacker et al.9, Mori et al.10 and

3 Brown, H.J. et al. (1993) Three-dimensional structure of the human class II histocompatibility antigen HLA-DR. Nature 364, 33–39 4 Adorini, L. et al. (1988) In vivo competition between self peptides and foreign antigens in T cell activation. Nature 334, 623–625 5 Hurtenbach, U. et al. (1993). Prevention of autoimmune diabetes in non-obese diabetic mice by treatment with a class II major histocompatibility complex-blocking peptide. J. Exp. Med. 177, 1499–1504 6 Woulfe, S.L. et al. (1997). A peptidomimetic which specifically inhibits human leukocyte antigen DR4 Dw4-restricted T cell proliferation. J. Pharmacol. Exp. Ther. 281, 663–669 7 Todd, J.A. et al. (1988) A molecular basis of MHC class II associated autoimmunity. Science 240, 1003–1009 8 Dessen, A. et al. (1997) X-ray crystal structure of HLA-DR4 complexed with a peptide from human collagen II. Immunity 7, 473–481 9 Hammer, J. et al. (1995). Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association. J. Exp. Med. 181, 1847–1855 10 Stern, L.J. et al. (1994). Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature, 368, 215–221 11 Hammer, J. et al. (1994). High affinity binding of short peptides to MHC class II molecules by anchor combinations. Proc. Natl. Acad. Sci. U. S. A. 91, 4456–4460

Liu et al.11, also reported the action of U-II from various species on rat GPR14 (SENR). Importantly, two of these groups9,10 attempted to identify the endogenous ligand from extracts of mammalian tissues that were tested for endogenous activity using Chinese hamster ovary cells expressing the orphan receptors. Mori et al. identified the sequence of two isoforms of porcine U-II (differing only by a proline for threonine substitution at the third residue) by amino-acid analysis of extracts from 50 spinal cords. Both peptides retained the cyclohexapeptide sequence of human U-II (Ref. 10). The cloning of preproU-II has recently been reported in a further two mammalian species; the cyclic region that is thought to be important in ligand binding is also conserved in the deduced amino acid sequence of rat and mouse U-II, whereas the N-terminal is more divergent from the human sequence12. These studies provide further validation of the success of two distinct strategies, screening either known agonists or tissue homogenates, to identify endogenous ligands for orphan receptors.

0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

PII: S0165-6147(00)01449-8

COMMENT Human UT-II receptor

U-II peptide

The deduced sequence of human GPR14 (UT-II receptor) comprises 389 amino acids5 with few surprises. Like many other GPCRs, cysteine residues are present in the first and second extracellular loops and are thought to be crucial in achieving the correct tertiary structure. The receptor has two potential glycosylation sites in the N-terminal domain, Asn29 and Asn33 (usually important in positioning the receptor within the cell), and two cysteine residues in the C-terminal with the potential for post-translational addition of palmitate. Binding of U-II to the human GPR14 receptor was selective5. The somatostatin receptor sst4 displayed the closest (albeit modest) amino acid sequence similarity, with 41% in the transmembrane domains and ~27% overall. Somatostatin-1–14 is thought to be one of the endogenous ligands for the sst4 receptor. Although somatostatin-1–14 shares some structural similarity with U-II, this peptide did not compete at a high (1 mM) concentration. Urotensin I (a 41 amino acid vasodilator peptide also isolated from the teleost urophysis) and other vasoactive peptides including neuropeptide Y and angiotensin II did not compete for binding, although endothelin-1 was not tested5. Iodinated goby U-II bound with sub-nanomolar affinity (Kd 5 0.4 nM) to expressed human GPR14 receptors; human U-II competed for this binding with a Ki of 2 nM with Hill slopes close to one. The authors did not report any evidence of further subtypes5. The density of receptors measured in rat arterial membranes was comparatively low (2–20 fmol mg21 protein) although these values are similar to Bmax values for endothelin-1 receptors in human arteries13. mRNA for human GPR14 was widely expressed in human tissues, including the left atrium and ventricle from the heart and, as expected, smooth muscle cells from the coronary artery and aorta. Intriguingly, mRNA was also detected in endothelial cells from several vascular beds5, which is in agreement with previous observations that goby U-II causes endothelium-dependent relaxation of rat aorta (by possibly releasing nitric oxide), although the predominant action is vasoconstriction4.

Surprisingly, the distribution of U-II mRNA in human CNS tissues5,8,9 was restricted to the medulla oblongata of the brain (the major integrating centre for blood pressure control) and spinal cord, with U-II-like immunoreactivity localized to motor neurones of the ventral horn. A broadly similar distribution of U-II mRNA was displayed in the CNS of rats and mice with the most intense hybridization signals also present in the medulla oblongata and spinal cord12. Although U-II-like immunoreactivity was reported in the vasculature with diffuse staining in the heart, U-II mRNA was not detected in cardiovascular tissues including the heart5, which expresses comparatively high levels of mRNA for the GPR14 receptor. It is unclear whether the concentration of mRNA is simply below the level for detection or whether these results suggest that human U-II might circulate in the plasma (rather than be released locally) and might be synthesized some distance from target receptors. The functional morphology of the teleost urophysis is similar to the neurohypophysis of the pituitary, and could be one candidate for the source of the peptide, although this endocrine gland was not examined for expression of U-II. Coulouarn et al.8 also reported highest expression of U-II mRNA in human spinal cord but found a more widespread distribution (by dot blot analysis), of human prepro-U-II mRNA in peripheral tissues, including the adrenal glands, kidney and spleen, which is suggestive of local synthesis and release in peripheral tissues. By contrast, Nothacker et al., using similar techniques, found the highest expression of human prepro-U-II mRNA in human kidney, whereas all other peripheral tissues examined expressed little or no precursor9. Further studies are needed to clarify the cellular origin of the endogenous peptide and resolve these discrepancies. Functional studies

Unexpectedly, Ames et al. found that vasoconstrictor activity of human U-II in isolated vessels from the rat was limited to the thoracic aorta; the abdominal aorta, and renal and femoral arteries did not respond even though goby U-II

(a) Goby

Val Cys Tyr Lys Ala Gly Thr Ala Asp Cys Phe

(b) Human

Trp

Val Cys Tyr Lys Glu Thr Pro Asp Cys Phe

(c) Porcine

Trp

Val Cys Tyr Lys

Gly Pro Thr Ser Glu Cys Phe

(d) Mouse

Trp

Ile Cys Tyr Lys

Gln His Gly Ala Ala Pro Glu Cys Phe

Trp

trends in Pharmacological Sciences

Fig. 1. Amino-acid sequences for (a) goby1, (b) human8, (c) porcine10, and (d) the predicted sequence for mouse, urotensin II (U-II)12. A second isoform of porcine U-II, differing only by a proline for threonine substitution at the third residue has also been identified10. The deduced sequence of rat U-II differs from the mouse by a threonine for alanine substitution in the fourth residue12. Shading indicates the C-terminal cyclic hexapeptide sequence, which is conserved across species.

contracted these and other vessels from the rat2. Thus, there might be a subtle difference in the responsiveness of rat receptors to both human and goby U-II. In contrast to the limited action in the rat, human U-II potently constricted all isolated vessels tested from cynomolgus monkeys, including coronary arteries, with EC50 values more potent (by about an order of magnitude) than those obtained for endothelin-1. The constrictor response developed very slowly, reaching a maximum at each concentration tested after 45–60 min5. This constriction is slower than that induced by endothelin-1, which would typically plateau within 30 min or less in human vessels in vitro. U-II-mediated vasoconstriction in monkeys was limited to the arterial side. By contrast, we have found that U-II constricts several human vessels including saphenous veins and coronary arteries.

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COMMENT (Patho)physiological role?

Acknowledgements We thank the British Heart Foundation for support.

Perhaps the most remarkable discovery of the report by Ames et al. was that systemic administration of human U-II to anaesthetized monkeys resulted in a dramatic and fatal cardiovascular collapse5. Human U-II, at doses of .30 pmol kg21, severely depressed myocardial contractility, and septal and free-wall motion was almost completely abolished. Significantly, ST segment changes in the electrocardiogram were typical of those seen in myocardial ischaemia, consistent with constriction of the coronary arteries. Although the precise physiological role of U-II in humans remains to be elucidated, the authors speculate that the results from the functional studies suggest that the peptide might modulate cardiovascular homeostasis in the periphery. The presence of immunoreactivity in the spinal cord motoneurones implies U-II might have additional roles, possibly influencing activity in the CNS. The marked increase in peripheral resistance in monkeys combined with an extraordinary reduction in cardiac contractility provide both a tantalizing prospect

for a role of U-II in heart failure and a stimulus to further research. With over 80 orphan GPCRs still awaiting identification of a ligand and perhaps another 5000 to be discovered in the human genome14, this research has wider significance, reinforcing the importance of pairing ligands with orphan receptors in the discovery of novel therapeutic targets. Selected references 1 Bern, H.A. et al. (1995) Neurohormones from fish tails: the caudal neurosecretory system. I. ‘Urophysiology’ and the caudal neurosecretory system of fishes. Recent Prog. Horm. Res. 41, 533–552 2 Itoh, H. et al. (1987) Contraction of major artery segments of rat by fish neuropeptide urotensin II. Am. J. Physiol. 252, R361–366 3 Itoh, H. et al. (1988) Functional receptors for fish neuropeptide urotensin II in major rat arteries. Eur. J. Pharmacol. 27, 149, 61–66 4 Gibson, A. (1987) Complex effects of Gillichthys urotensin II on rat aortic strips. Br. J. Pharmacol. 91, 205–212 5 Ames, R.S. et al. (1999) Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 401, 282–286 6 Marchese, A. et al. (1995) Cloning and chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8, neuropeptide Y, and somatostatin receptors. Genomics 29, 335–344

7 Tal, M. et al. (1995) A novel putative neuropeptide receptor expressed in neural tissue, including sensory epithelia. Biochem. Biophys. Res. Commun. 209, 752–759 8 Coulouarn, Y. et al. (1998) Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord. Proc. Natl. Acad. Sci. U. S. A. 95, 15803–15808 9 Nothacker, H.P. et al. (1999) Identification of the natural ligand of an orphan G-proteincoupled receptor involved in the regulation of vasoconstriction. Nat. Cell Biol. 1, 383–385 10 Mori, M. et al. (1999) Urotensin II is the endogenous ligand of a G-protein-coupled orphan receptor, SENR (GPR14). Biochem. Biophys. Res. Commun. 265, 123–129 11 Liu, Q.Y. et al. (1999) Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14. Biochem. Biophys. Res. Commun. 266, 174–178 12 Coulouarn, Y. et al. (1999) Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors. FEBS Lett. 457, 28–32 13 Davenport, A.P. et al. (1995) Endothelin ETA and ETB mRNA and receptors expressed by smooth muscle in the human vasculature: majority of the ETA sub-type. Br. J. Pharmacol. 114, 1110–1116 14 Marchese, A. et al. (1999) Novel GPCRs and their endogenous ligands: expanding the boundaries of physiology and pharmacology. Trends Pharmacol. Sci. 20, 370–375

LETTERS

G-protein-coupled receptors: what limits high-affinity agonist binding? In a recent review1, Roland Seifert et al. described the utility of G-proteincoupled receptor (GPCR)–G-protein fusion proteins for studying receptor– G-protein coupling. Among the many insights provided, a surprising observation was noted. Despite the defined 1:1 stoichiometry of the receptor and G protein, only a fraction of the total receptor population displays high affinity for agonist ligands, which is characteristic of the ternary complex state of the receptor, agonist and G protein. The same discrepancy has been observed in native (non-fused) systems in which the G protein is in large excess over the receptor2. With respect to the fused system, possible explanations are discussed by Seifert et al. – not all of the fused G protein might be functional or the

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ternary complex might not be stable1. In non-fused systems, proposed explanations include compartmentalization of the receptor and G protein2 and receptor dimerization3. An alternative or additional explanation can be proposed for both systems on the basis of the known properties of receptors and G proteins. High-affinity agonist binding is measured in radioligand binding assays that employ cell membrane preparations that have been washed to remove guanine nucleotide. The G protein in the agonist high-affinity ternary complex is in the trimeric form (a-subunit coupled to the bg-dimer). GDP strongly stabilizes the trimeric form of the G protein. In binding assays, the concentration of free GDP could be low enough to limit the association of the G-protein a-sub-

unit and bg-dimer so that only a fraction of the G protein is in a form that can interact with the receptor to form the high-affinity agonist binding state. Typically, G proteins are in considerable stoichiometric excess over the receptors to which they couple2,4. In studies of the b2-adrenoceptor and Gs, the G-protein concentration in the membrane has been shown to be one to two orders of magnitude greater than that of the receptor4. The G protein in the ternary complex state is believed to be in the trimeric form (Fig. 1) – both the a-subunit and bg-dimer are required to reconstitute high-affinity agonist binding5,6. The guanine-nucleotide-binding site on the a-subunit is believed to be empty within this complex (Gae.bg) (Fig. 1)6. The association of the a-subunit with the bg-dimer is regulated by the agonistoccupied receptor (Fig. 1) and by GDP. The activated receptor stabilizes the trimeric G protein by binding to it with