Cloning and characterization of corticotropin-releasing factor and urocortin in Syrian hamster (Mesocricetus auratus)

Peptides 20 (1999) 1177–1185

Cloning and characterization of corticotropin-releasing factor and urocortin in Syrian hamster (Mesocricetus auratus) B.M. Robinsona,1, D.J. Tellama,1, D. Smartb, Y.N. Mohammada, J. Brennandc, J.E. Rivierd, D.A. Lovejoya,* a

University of Manchester, School of Biological Sciences, 3.614 Stopford Building, Manchester, M13 9PT, UK Parke Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge, CB2 2QB, UK c Zeneca Pharmaceuticals, Mereside Alderly Park, Macclesfield, Cheshire, UK d Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA b

Received 11 February 1999; accepted 25 May 1999

Abstract Corticotropin-releasing factor and urocortin belong to a superfamily of neuropeptides that includes the urotensins-I in fishes and the insect diuretic peptides. Sequence analysis suggests that urocortin is the mammalian ortholog of urotensin-I, although the physiological role for this peptide in mammals is not known. Within the Rodentia, hamsters belong to a phylogenetically older lineage than that of mice and rats and possess significant differences in hypothalamic organization. We have, therefore, cloned the coding region of the Syrian hamster (Mesocricetus auratus) corticotropin-releasing factor and urocortin mature peptide by polymerase chain reaction. Hamster urocortin was prepared by solid-phase synthesis, and its pharmacological actions on human corticotropin-releasing factor R1 and R2 receptors were investigated. The deduced hamster corticotropin-releasing factor amino acid sequence and cleavage site is identical to that in rat, whereas the urocortin sequence is unique among the urocortin/urotensin-I/sauvagine family in possessing asparagine and alanine in positions 38 and 39, respectively. The hamster urocortin carboxy terminus sequence bears greater structural similarity to the insect diuretic peptide family, suggesting either retrogressive mutational changes within the mature peptide or convergent sequence evolution. Despite these changes, human and hamster urocortin are generally equipotent at cAMP activation, neuronal acidification rate, and R1/R2 receptor affinities. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Stress; Primary structure; Peptide evolution; Receptor-ligand binding

1. Introduction Since the publication of the structural characterization of corticotropin-releasing factor (CRF) by Vale and his associates in 1981 [34], CRF remains the predominant hypothalamic neuropeptide regulating renal glucocorticoid release via pituitary adrenocorticotropic hormone release in vertebrates. Outside of the paraventricular nucleus, CRF-producing neurons are found throughout the neocortex, hippocampus, and septal regions of the forebrain, as well as within discrete cell groups within the midbrain and hindbrain [27]. * Corresponding author. Tel.: ⫹44-161-275-3881; fax: ⫹44-161-2753938. E-mail address: [email protected] (D.A. Lovejoy) 1 B.M. Robinson and D.J. Tellam should be regarded as joint first authors of this paper.

In these regions, CRF appears to function as a neurotransmitter or modulator. Changes in CRF regulation have been implicated with appetite and metabolic disorders, neurodegeneration, and reproductive dysfunction [2,16,24,31,39], although a clear cause-and-effect relationship has yet to be established. In fishes, urotensin-I, produced in the caudal neurosecretory system (urophysis), stimulates the release of glucocorticoids directly from the interrenal gland, apparently bypassing the pituitary [15]. Urotensin-I, originally characterized by Lederis and his associates in 1982 [12], is a structural homolog of CRF. In 1995, we reported the cloning and characterization of an apparent rat ortholog of urotensin-I, urocortin, expressed in the mid- and hindbrain of rat [35]. Recent studies suggest that the neural expression of this peptide may be much wider than previously thought and, therefore, may be involved in several neural systems [36,

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38]. Urocortin possesses only ⬇50% sequence identity with rat/human CRF but ⬇60% identity with carp and sucker urotensin-I. Human urocortin [5] shows a similar structural relationship. Thus, urocortin and its anuran homolog, sauvagine [20], appear to represent an orthologous tetrapod peptide lineage to the piscine urotensin-I. However, the function of urocortin in mammals remains a mystery as a functional homolog to the urophysis has never been identified. Although urocortin recently was reported to possess greater potency and specificity than CRF in the inhibition of appetite [31], its primary sites of synthesis in the midbrain and hindbrain do not appear to be associated with appetite regulation per se. Because CRF and urocortin possess high affinity for both R1 and R2 receptors and the CRF-binding protein at physiological concentrations [31,35], in vivo physiological studies with the peptides produce many confounding observations. This problem may be solved in part by the development of alternate animal models. The Syrian hamster belongs to a phylogenetically older lineage of rodents than that of the rats and mice [6,7,9] and possesses significant differences in the neural organisation of CRF [3]. An investigation of hamster urocortin and CRF may provide some insight into the phylogenetic and physiological constraints acting to drive the evolution of these peptides. We have, therefore, cloned and sequenced the active peptide portions of hamster CRF and urocortin.

2. Methods 2.1. Animals and tissue dissection Adult golden hamsters (Mesocricetus auratus) of mixed sex were euthanized by cerebral dislocation. Small (0.5–1.5 g) portions of the liver were removed and quickly frozen by immersion in liquid nitrogen. The frozen tissue was stored at ⫺70°C for ⬇2–3 months before extraction.

2.3. Polymerase chain reaction (PCR) Nested-primer PCR was used to amplify both CRF and urocortin gene fragments. All primers were prepared at the University of Manchester oligonucleotide synthesis facility (see Fig. 1). The outside CRF primers were 5⬘-ATTCT GATCCGCATGGGTGAAGAATACTTC-3⬘ and 5⬘-TA ATTAGGGGTATATAGGCTCTCTCCCTG-3⬘ for the sense and antisense positions, respectively. The sense and antisense second-round nested primers consisted of 5⬘AACTTTTTCCGCGTGTTGCTGCAGCGCAGCTG-3⬘ and 5⬘-TGCAGAATCGTTTTGGCCAAGCGCAACATT-3⬘, respectively. The outside primer reactions contained 50 ␮M each primer, 50 ␮M each dNTP, 1–3 ␮g of hamster genomic DNA, with a MgCl2 concentration of 2.0 mM at pH 9.0. The inside reactions were the similar but used 2– 4 ␮l of the first reaction instead of genomic DNA. PCR products were resolved on a 1.5% low melting temperature agarose (FMC) gel. The bands corresponding to the correct sizes were excised and eluted by freezing the gel pieces in the presence of equilibrated phenol. The amplified DNA was reprecipitated in 3 M sodium acetate and 100% ethanol, dried, and resuspended in 5 ␮l of Milli-Q (Pharmacia) water. All fragments were subcloned into a pGEM-T vector by using JM106 cells as a host strain (Promega, Madison, USA). Clones possessing the insert were confirmed by restriction digest of mini-prep purified DNA. The clones of interest were amplified by culture in LB medium and purification using a Qiagen (Chatsworth, CA, USA) ‘Maxi Plasmid Kit.’ The subcloned fragments were sequenced by standard dideoxy chain termination (Sequenase, U.S. Biochemicals, Cleveland, OH, USA). Due to the incidence of potential errors in the DNA amplification process by PCR, the hamster cDNA fragments were amplified by PCR a second time using the outer primers to confirm the original sequence. The amplified fragments were subcloned and sequenced in the same manner as the first. 2.4. Peptide synthesis

2.2. DNA extraction Genomic DNA was extracted by first pulverizing the frozen tissue in liquid nitrogen then transferring it to a solution containing 10 mM Tris䡠HCl, 0.1 M ethylenediaminetetraacetic acid, 20 ␮g/ml pancreatic RNase, and 0.5% sodium dodecyl sulfate in TE buffer (pH 8.0) before agitating for 60 min at 37°C. Proteinase K (20 ␮g/␮l) was added to the tissue solution, mixed, and then transferred to a 50°C water bath with gentle agitation. Undigested material was removed by centrifugation (3000 ⫻ g) for 10 min. The proteinaceous material was removed with two extractions with equilibrated phenol. DNA was precipitated in 2 vol of 100% ethanol and 0.1 vol of 3 M sodium acetate. After centifugation at 3000 ⫻ g, the pellet was washed in 70% ethanol, dried briefly, and resuspended in 1 ml of TE buffer (pH 8.0).

Hamster urocortin, based on the deduced primary structure from the cDNA sequence, was prepared by solid-phase synthesis and was purified as previously described [11]. The methylbenzhydrylamine resin was prepared by using methods developed by Rivier et al. [26]. Human urocortin and rat CRF were obtained from commercial sources. 2.5. CRF receptor binding assay Radiolabeled (125I) urocortin was used as the competitor for CRF-R2, and 125I-CRF was used for CRF-R1 transfected membranes. Unlabeled hamster urocortin was serially diluted in indicator-free Dulbecco’s modified Eagle’s medium (DMEM; 14 dilutions; range 3– 0.1 nM) then dispensed in triplicate into 96-well plates. Radioactive 125I-urocortin and 125 I-CRF then were diluted to 0.002 ␮Ci/␮l in 1⫻ mem-

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Fig. 1. Sequence analysis of hamster CRF. (A) The total length of the CRF fragment was 565 bp. The boxed region indicates position of the mature peptide in the cloned fragment. (B) Sequence of fragment. The shaded regions indicate the positions where the PCR primers were expected to hybridize. The bold letters indicate the position of the mature peptide. The boxed regions at the beginning and end of the mature peptide indicate the cleavage and amidation motifs, respectively.

brane buffer (50 mM Tris, 10 mM MgCl2, and 2 mM ethylenediaminetetraacetic acid, pH 7.0), and 10 ␮l was added to the appropriate replicates. CRF-R1- and CRF-R2transfected membranes then were diluted in 1⫻ membrane buffer to 0.2 ␮g/␮l and 0.3 ␮g/␮l, respectively. An 80-␮l aliquot of the scintillation proximity assay (Amersham, Little Chalfont, UK)-bead/receptor-membrane mix (20-␮l wheatgerm agglutinin scintillation proximity assay beads in 1⫻ bead buffer; 10 ␮l CRF-R1/R2 membranes in 1⫻ membrane buffer; 8 ␮l of 10⫻ membrane buffer; 10 ␮l of 10% dimethyl sulfoxide; and 32 ␮l of H2O) then was added to each well; 96-well plates then were sealed and incubated overnight at room temperature. Differences between replicates were measured by using a topcounter.

cells were subsequently cotransfected with a ␤-galactosidase-LacZ reporter gene (Invitrogen) and grown to a density of 1.25 ⫻ 106 cells per ml in indicator-free DMEM (GIBCO, Paisley, UK). Aliquots of 50 ␮l were transferred to each well on 96-well plates. Sixteen dilutions of each peptide (range 0.002–1 mM) then were prepared in indicator-free DMEM. Fifty microliters of each of the diluted peptides were added in triplicate to the appropriate replicates of each dilution. Cells and ligands then were incubated for 5 h in 5% CO2. Fifty microliters of the 0.5 mM chlorophenol red/D-galactopyranoside indicator solution (Boehringer Mannheim, Mannheim, Germany) in a sodium phosphate buffer (pH 7.0) was added to each well. After 1–2 h, optical density was measured at 570 nm.

2.6. ␤-galactosidase reporter gene assay

2.7. Culture of CHO cells

Chinese hamster ovary (CHO) cells were transfected with the human R1 or R2-CRF receptor by using a pCMV expression vector (Invitrogen; Carlsbad, CA, USA). These

CHO-Pro5 cells routinely were grown as monolayers in MEM-Alpha medium supplemented with 10% fetal calf serum (GIBCO) and maintained under 5% CO2 at 37°C.

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Cells were passaged every 4 –5 days, and the highest passage number used was 16. Before use in the Cytosensor, the cells were seeded on the polycarbonate microporous membranes of the capsule cups (Molecular Devices, Crawley, UK) to achieve a density of ⬇0.6 million cells/cup on the day of the experiment. 2.8. Cellular acidification measurements A Cytosensor microphysiometer (Molecular Devices) was used to measure the extracellular acidification rate, as described previously [8,18]. CHO cells expressing recombinant human CRF-R1 or CRF-R2 receptors were seeded into Cytosensor capsule cups (see above) and placed in sensor chambers at 37°C inside the Cytosensor and maintained by a flow (120 ␮l/min) of bicarbonate-free Hams F-12 containing 0.2% bovine serum albumin (pH 7.4). The flow was halted for 22 s at the end of each 2-min pump cycle, and the rate of acidification (␮V/s) was measured for 15 s during that period. Hamster or human urocortin (0.3– 300 pM) was introduced sequentially in the perfusate for 10 min at 45-min intervals. CHO cells not transfected with the CRF receptors were used as negative controls.

the mature peptide is amidated. The amino acid sequence derived from the amplified portion of the preprohormone upstream of the mature peptide indicated 84% and 86% sequence identity to the rat and mouse, respectively. As expected, a reduced amino acid sequence identity of ⬇66% was obtained when compared to the human sequence. A 305-bp product was obtained by using the outer rat urocortin-based primers (Fig. 2). Reamplification by using the inner primers yielded a fragment of 171 bases. Sequence analysis of both fragments confirmed the hamster urocortin sequence including the cDNA spanning the 16 residues of the upstream preporhormone, the mature peptide with a glycine-lysine amidation motif, and 77 bases of the 3⬘ untranslated region. The deduced amino acid sequence of the amplified cDNA fragment indicates that the mature peptide possesses 88% and 82% sequence identity with the rat and human peptides, respectively (Fig. 3). Hamster urocortin differs from human urocortin in positions 1– 4, 37, 38, and 39. Compared to rat urocortin, positions 2 and 4 in the hamster ortholog are substituted, although positions 1 and 3 are the same. The cleavage site (RQRR) between the mature and cryptic peptides is identical to that in the rat peptide. A second round of PCR amplification yielded a fragment with a sequence identical to the first.

2.9. Data analysis 3.2. Receptor binding and activation Comparisons of the receptor binding and ␤-galactosidase activation data were assessed by using a one-way analysis of variance. Bonferroni’s multiple comparison test was used to determine the level of significance (established at P ⬍ 0.05) between data sets. Agonist effects with the Cytosensor were determined quantitatively as the increase in the acidification rate response (peak ⫺ basal) and are expressed as ␮V/s or as a percentage of the maximal response, as appropriate. Data are presented as mean ⫾ SEM (n ⫽ 3) unless otherwise stated. Statistical analysis, curve-fitting, and parameter estimation for all analyses were carried out by using Graphpad Prism 2.01 (San Diego, CA, USA).

3. Results 3.1. CRF and urocortin primary structure PCR amplification by using the outer CRF-based primers resulted in the expected-sized fragment of 565 bp. The sequence analysis of the subcloned fragment from the inner primers indicated a 162-bp fragment with a high sequence similarity to rat CRF. Both fragments were double-strand sequenced (Fig. 1). The obtained sequence corresponded to a region spanning the middle of the cryptic peptide in the 5⬘ region to 106 bases into the 3⬘ untranslated region. The amino acid sequence deduced from the cDNA sequence of the mature hamster CRF peptide and expected cleavage site is identical to that of rat and human. The CRF residue sequence terminates in a glycine-lysine pair, suggesting that

Hamster urocortin bound the CRF-R1 with an affinity of 14.3 ⫾ 1.2 nM (Fig. 4a) in transfected mouse erythroleukemia cells. This value was not significantly different from the human urocortin affinity (16.1 ⫾ 1.2 nM) but was significantly greater than that of rat/human/hamster CRF on the R1 receptor (54.7 ⫾ 1.1 nM; P ⬍ 0.001) by using a one-way analysis of variance with Bonferroni’s multiple comparison test. There were no significant differences between the EC50 of the human (1.4 ⫾ 1.5 nM) and hamster (2.9 ⫾ 1.3 nM) urocortins and rat CRF (2.5 ⫾ 1.3 nM) at eliciting ␤-galactosidase activity via the R1 receptor (Fig. 4b). Rat CRF and hamster urocortin bound the R2 receptor with a similar affinity (270.6 ⫾ 1.2 nM and 135.6 ⫾ 13.1 nM, respectively; Fig. 5a), whereas the human urocortin ligand bound the R2 receptor with an affinity of 370.6 ⫾ 1.2 nM. Analysis of variance indicated all were not significantly different. The EC50 for the hamster urocortin (0.7 ⫾ 1.2 nM) was also not significantly different to that of human urocortin (0.7 ⫾ 1.1 nM) although rat CRF (5.6 ⫾ 1.5 nM) produced a significantly (P ⬍ 0.001) lower activity with respect to ␤-galactosidase reporter activity via an R2-dependent mechanism (Fig. 5b). 3.3. Cytosensor microphysiometry Hamster urocortin increased the acidification rate in CHO cells expressing the human CRF-R1 or -R2 receptor, characterized at higher concentrations as a broad monopha-

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Fig. 2. Sequence analysis of hamster urocortin (A). The length of the urocortin fragment was 305 bp. The boxed region indicates the position of the mature peptide in the cloned fragment. (B) Sequence of fragment. The shaded portions indicated where the PCR primers were expected to hybridize. The bold letters indicate the position of the mature peptide. The boxed regions at the beginning and end of the mature peptide indicate the cleavage and amidation motifs, respectively.

sic response that peaked 10 –12 min after the onset of agonist perfusion and then returned slowly to baseline (Fig. 6). Human urocortin also elicited a similar acidification responses (Fig. 6). Moreover, the acidification response to both hamster or human urocortin was concentration-dependent (Fig. 6), with pEC50 values of 11.26 ⫾ 0.17 vs. 11.12 ⫾ 0.22 at the hCRF1 receptor and 11.50 ⫾ 0.03 vs. 11.42 ⫾ 0.02 at the hCRF2 receptor, respectively. Untransfected CHO cells were unresponsive to the presence of CRF or urocortin in the medium.

4. Discussion These data indicate that, although the primary structure of CRF has been conserved in the Syrian hamster (Mesocricetus auratus), urocortin has five substitutions relative to its murine ortholog. The sequence changes may reflect not only the phylogenetic position of hamsters within the

order Rodentia but also different adaptive pressures impinging upon the hamster urocortin system. However, despite these changes, synthetic hamster urocortin is equipotent to human urocortin and binds with a similar affinity to the human R1 and R2 receptors. These sequences can be used as probes to investigate the roles of stress and appetite regulation in the hamster lineage. The large number of residue substitutions in hamster urocortin, relative to that in rats and mice, is surprising. However, this may reflect, in part, the phylogenetic ancestory of this species. Myomorph rodents are a relatively heterogenous collection of animals for which a clear phylogenetic ancestry is unresolved [6,7,9]. Hamsters belong to the family Cricetidae, which is generally regarded as a sister group to the Muridae, which includes both rats and mice. Some studies, for example, ␣-1-antiproteinase sequence data, suggest that the hamsters may be a phylogenetically older lineage within the Cricetidae [22]. In addition, the neuroanatomical organization of the hamster paraventricular nucleus and immunoreactive CRF varies between the rat

Fig. 3. Comparison of the hamster, rat, and human urocortin sequences with carp urotensin-I (UI) and tobacco hornworm (Manduca sexta) diuretic peptide. The shaded portions indicate the residue substitutions relative to rat urocortin. The clear boxed regions indicate residues in common with the M. sexta peptide.

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Fig. 4. Receptor binding affinity (a) and ␤-galactosidase activity (b) in R1-transfected cells. Mouse erythroleukemia cells were used for the receptor binding assay, whereas CHO cells were used in the ␤-galactosidase assay. The synthetic hamster and human urocortins possessed statistically similar affinties.

and hamster [3,21]. Thus, the Cricetidae and Muridae may be more divergent than previously thought. The hamster CRF sequence is identical to that found in nonruminant mammals. The mature CRF peptide is identical in rats, mice, dogs, horses, and humans [13,19,28] and would not be expected to be different in hamsters. A unique set of evolutionary pressures has apparently driven the ruminant (bovine, ovine, caprine) CRF sequence in a different direction [14]. The partial sequence of the hamster CRF cryptic peptide shows a similar number of substitution events relative to both rats and mice (84% and 86%, respectively). Over the same sequence interval, rats and mice possess 91% residue identity relative to each other. In contrast, there were five amino acid substitutions in the hamster urocortin sequence, although all were conservative and are found in other CRF superfamily members (Fig. 3). Within the amino acid sequence of CRF, urotensin-I, and insect diuretic peptides, the terminal residues show the greatest variation. The significance of the leucineasparagine-alanine (L-N-A) substitution of phenylalanineaspartic acid-serine (F-D-S) at the carboxy terminus (Fig. 3) in comparison to human and rat urocortin is unclear. A leucine-asparagine motif is also present in the homologous region of Manduca sexta diuretic peptide [10], a peptide

Fig. 5. Receptor binding affinity (a) and ␤-galactosidase activity (b) in R2-transfected cells. Mouse erythroleukemia cells were used for the receptor binding assay, whereas CHO cells were used in the ␤-galactosidase assay. The synthetic hamster and human urocortins possessed statistically similar potencies.

distantly related to vertebrate CRFs. However, this motif is not present in other urocortin or CRF sequences from species phylogenetically older than hamsters. This suggests that the presence of this motif in hamster urocortin represents a retrogressive mutation to that of an ancestral condition. Alternately, its presence in the homologous region of hamster urocortin and M. sexta diuretic peptide may indicate convergent evolution. In any case, the occurrence of either is rare, thus the probablity of such an event would increase if structural constraints imposed upon the peptide by the receptor and processing enzymes reduced the number of available residues to be functional at this locus. In ovine CRF, however, substitution of the 38, 39, or 40th residue with alanine produces a significant change in potency as defined by adrenocorticotropic hormone release from rat anterior pituitary cells [11], suggesting that these residues are important on the R1 receptor. The primary structure of a polypeptide hormone can be divided into a series of functionally discrete stretches of amino acids ⬇5–20 residues long [14,16]. The CRF subfamily of peptides could be divided into four such ‘microdomains’: a nonfunctional amino terminus (residues 1– 4), a conserved R1 activation region, and proteolytic sites (resi-

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Fig. 6. Hamster or human urocortin elicits a concentration-dependent acidification response in CHO cells expressing the human CRF1 (A) or CRF2 (B) receptor. Upper panels depict the concentration-response curves for hamster (n) or human (F) urocortin (mean ⫾ SEM, n ⫽ 3), whereas the lower panels depict representative Cytosensor traces for hamster urocortin at the CRF1 (A) and CRF2 (B) receptor.

dues 5–16), an ␣-helical internal portion that includes the binding protein [13,31] binding region (residues 17–33), and a carboxy terminal region (residues 34 – 41). With the urocortin/urotensin-I carboxy terminal region, two directions of evolution can be discerned. Among the sarcopterygian or lobe-finned fish lineage, ultimately leading to tetrapods, this sequence is N-R-I/L-I/L-F/L-D-S-V. For the equivalent region among the actinopterygian lineage (rayfinned fishes), the sequence is N-R-R/K/N-I/Y/L-D-E-V. Its conservation within the respective lineages indicates a physiological constraint to retain the sequence. The hamster carboxy terminal sequence, therefore, is unique. The pharmacological data presented in this paper indicates that, despite the residue substitutions, there are no apparent effects on receptor affinity or cAMP activation as defined by our ␤-galactosidase assay. However, it is unclear whether this pharmacological similarity would occur in vivo. We have used a heterologous assay system in which human R1 and R2 receptors were transfected into mouse erythroleukemia cells. In cell lines naturally expressing the receptor, the affinities of the R2 receptor for its ligand relative to the R1 receptor tend to be higher [5,35]. In our systems, lower affinities were observed with the R2 receptors, similar to that previously noted by Liaw and associates [17]. Thus, these differences may reflect, in part, transfec-

tion efficiencies. Moreover, Xiong and colleagues [37] noted that human R2-transfected COS-7 cells produce significantly lower levels of cAMP, an effect they attribute to lower adenylate cyclase coupling ability by the R2 receptor. The Cytosensor microphysiometer is a device that quantifies cellular metabolic activity by measuring the changes in the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation [23,30]. The acidification rate is measured as the change in potential across a silicon light-addressable sensor, caused by the accumulation of hydrogen ions during periods of cessation of the flow of medium [18]. Thus, this method is particularly appropriate for examining the activation of a cell by a particular stimulus. The hamster urocortin response was indistinguishable from that of the human urocortin response, indicating that despite the seven residue substitutions, they were not significant in regulating the activation of these transfected CHO cells.

Acknowledgments We thank S. Lahrichi and C. Miller (The Salk Institute) for synthesis and purification of the hamster urocortin, A. Davies (Zeneca Pharmaceuticals) for assistance with the

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receptor assays, and Prof. A. Loudon and Dr A. Stirland for help with the collection and preparation of the hamster tissue. This work was completed with financial assistance from MRC Studentship (B.M. Robinson), BBSRC-CASE Studentship (Zeneca Pharmaceuticals) to D.J.T. (National Institutes of Health Grant DK-26741) to J.E.R., and a Wellcome Trust Project grant to D.A.L.

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