Rattus norvegicus melanocortin 3 receptor: A corrected sequence

Peptides 26 (2005) 1835–1841

Rattus norvegicus melanocortin 3 receptor: A corrected sequence Derek Daniels a,∗ , Aae Suzuki a , Edan Shapiro c , Laiyi Luo a , Daniel K. Yee a , Steven J. Fluharty a,b,c,d a

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, 220E, Philadelphia, PA 19104, USA b Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA c Biological Basis of Behavior Program, University of Pennsylvania, Philadelphia, PA 19104, USA d The Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA Received 16 July 2004; accepted 22 November 2004 Available online 27 June 2005

Abstract Examination of the Rattus norvegicus genome reveals differences in the melanocortin 3 receptor (MC3R) compared with the published sequence (accession X70667). To clarify these differences, we used RT-PCR to clone MC3R from Sprague Dawley rats. These efforts revealed a sequence for the rat MC3R consistent with that predicted by the rat genome, but different from the published receptor by three amino acids, all of which were located in the predicted second transmembrane domain (TM2). Analysis of these residues revealed that TM2 of the rat MC3R is more homologous with other species than previously considered. The presently described sequence maps onto chromosome 3 of the rat genome, which shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20. Transient expression revealed high affinity binding of [125 I]-NDP-MSH and a concentration-dependent cAMP response to the synthetic agonist MTII. These data both clarify the sequence of the MC3R and demonstrate the great utility of genomic information recently made available. © 2005 Elsevier Inc. All rights reserved. Keywords: Melanocortin receptor; Melanocyte stimulating hormone; MTII; cAMP

1. Introduction The central melanocortin receptors, melanocortin 3 receptors (MC3R) and melanocortin 4 receptors (MC4R), have been implicated in the control of energy homeostasis. MC4R mutation or deletion in humans [4,20,24,26,30] or mice [13,19] is associated with an obese, hyperphagic and diabetic phenotype. Compared to that of the MC4R, the role of MC3R in such regulatory functions is poorly understood. Previous experiments have shown differences in the anatomical localization of these receptor subtypes in the brains of rats and mice [3,15,21,23]. Moreover, the co-localization of MC3R, but not MC4R, with the endogenous ligands ␥melanocyte stimulating hormone (␥-MSH) or agouti related protein (AgRP) suggests that MC3R may have autoreceptor∗

Corresponding author. Tel.: +1 215 898 9149; fax: +1 215 573 5186. E-mail address: [email protected] (D. Daniels).

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.11.032

like functions, while MC4R serves primarily as a heteroreceptor [3]. Recent studies using in vitro models of these receptors have also revealed different signal transduction capacities of the mouse MC3R and the rat MC4R [9], although within species comparisons were not examined. More specifically, cAMP production by these receptors appears to be ubiquitous, while activation of MAP kinase may be limited to MC4R activation. MC3R-deficient (Mc3r-/- ) mice, like those deficient of MC4R, have increased adiposity accompanied by increased plasma leptin [5,6]. Male, but not female Mc3r-/mice display increased insulin, while female, but not male Mc3r-/- mice, exhibit increased motor activity compared to wild-type littermates [6]. Furthermore, an obesity quantitative trait locus has been identified in the region of the human chromosome 20 that contains the gene encoding MC3R [16]. Other studies of Mc3r variants, however, have not revealed a link with obesity [27] and attempts to attenuate food intake by selective activation of MC3R in rats have likewise failed

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[1]. Nevertheless, given the clear obese phenotype of Mc3r-/mice and the effect of drugs that act at both MC3R and MC4R, the need for a better understanding of these receptor subtypes is clear. Efforts to describe the Rattus norvegicus genome have been published recently [12] and these data are accessible in many formats including a BLAST search through the National Center for Biotechnology Information (NCBI). A comparison of the coding region of the previously published rat MC3R (GenBankTM accession X70667 [23]) with the rat genome revealed subtle differences in the nucleotide sequences that correspond to the substitution of three amino acids. To further examine these discrepancies, we used PCRbased techniques to directly clone the MC3R from the Sprague Dawley strain of R. norvegicus. The present report describes the nucleotide and predicted amino acid sequence of this receptor and provides binding and functional data for a corrected sequence of the R. norvegicus MC3R.

was used for the PCR with the first round primers 5 CAGCATCCACAAGAGAAGCA-3 in the forward direction and 5 -CTGAGCATTGCTTTTCTCTCTTG-3 in the reverse direction and the second round primers 5 -ATCCACAAGAGAAGCACCTAGAAG-3 forward and 5 -AGCCGTGGAACACCTCCT-3 reverse. These primers were designed using Primer3 (Whitehead Institute for Biomedical Research, Cambridge, MA [25]). Several PCRs were conducted on cDNA from each brain using either the Titanium Taq PCR kit (BD Biosciences Clontech, Palo Alto, CA), AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) or SuperTaq Polymerase (Ambion, Austin, TX). All PCR conditions were 30 cycles of 95 ◦ C (1 min), 50–60 ◦ C (1 min) and 72 ◦ C (1 min), followed by a 10 min extension step at 72 ◦ C. The resultant PCR products were subcloned into the vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA) for subsequent propagation and DNA sequencing performed by the DNA Core Facility in the University of Pennsylvania School of Veterinary Medicine.

2. Methods

2.4. Cell culture and transfections

2.1. Peptides

COS-1 cells were grown in polystyrene tissue culture flasks in medium consisting of D-MEM (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), l-glutamine and penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% O2 at 37 ◦ C. The newly cloned R. norvegicus MC3R cDNA was subcloned into the expression vector pCR3 (Invitrogen) and then introduced into the COS-1 cells by transfection with LipofectAMINE (Gibco) for 5 h, after which the transfection medium was removed and replaced with normal growth medium. Forty-eight to 72 h post-transfection, cells were harvested for binding experiments or used for functional assays (see below).

Iodinated (Nle4 ,D-Phe7 )-␣-MSH (NDP-MSH) was purchased from the Peptide Radioiodination Service Center of the University of Mississippi (University, MS). Unlabeled NDP-MSH, ␣-MSH, ␥1 -MSH, MTII and SHU9119 were purchased from Bachem (King of Prussia, PA). 2.2. Animals and tissue preparation Two adult male rats of the Sprague Dawley strain were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in plastic cages in a temperaturecontrolled room (22 ◦ C) with a 12-h light:12-h dark cycle and food and water available ad libitum. The handling and care of experimental animals conformed to the regulations provided by the NIH Guide for the Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. The rats were anaesthetized using ketamine and xylazine (90 and 12 mg/kg, respectively) and killed by cervical dislocation. The brains were rapidly removed from the crania and a block of tissue containing the hypothalamus, thalamus and septum was isolated. The tissue was submerged in RNALater (Ambion, Austin, TX) overnight at 4 ◦ C before RNA isolation using the RNeasy lipid tissue kit following product instructions (Qiagen Inc., Valencia, CA). 2.3. Cloning of MC3R First strand synthesis was performed using the First Strand cDNA Synthesis kit (Amersham Biosciences, Piscataway, NJ) with random primers. A nested approach

2.5. Membrane preparations Two days after transfection, cell membranes were prepared from the COS-1 cells. First, growth medium was removed from culture dishes, and the cells were rinsed two times in ice-cold 20 mM Tris–HCl (pH 7.4) and 150 mM NaCl. Cells then were incubated for 10 min at 4 ◦ C in 20 mM Tris–HCl (pH 7.4), after which they were scraped from the plate and lysed by polytron homogenization. Following this, the cells were centrifuged and the pellet was resuspended in 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2 and 1.5 mM CaCl2 . The protein concentration was determined by BCA protein assay (Pierce, Rockford, IL). The solution was then subject to a second centrifugation. The pellet was homogenized, as above, and resuspended to a protein concentration of 1 mg/ml in assay buffer, a solution of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2 , 1.5 mM CaCl2 , 0.2% heat-inactivated BSA, 0.3 TIU/ml aprotinin and 100 ␮g/ml 1,10-phenanthroline. The membrane solution was aliquoted and stored at −80 ◦ C.

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2.6. Radioligand binding assays The binding assays were initiated by the addition of 10 ␮g of diluted membrane protein to assay buffer containing various concentrations of [125 I]-NDP-MSH and unlabeled competitors. Non-specific binding was defined as the amount of radioligand binding remaining in the presence of 1 ␮M NDPMSH. Saturation isotherms used at least six concentrations of [125 I]-NDP-MSH, ranging from 0.6 to 3.15 nM. Competition binding analyses were performed using 0.32 nM [125 I]-NDPMSH for five unlabeled competitors: NDP-MSH, ␣-MSH, ␥1 -MSH, MTII and SHU9119. Eight concentrations of each unlabeled competitor ranging from 10−12 to 10−5 M were used in the analyses. The incubations proceeded for 120 min at 37 ◦ C, while shaking, and were terminated by rapid dilution with 5 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1.5 mM CaCl2 and vacuum filtration on glass-fiber filters presoaked with 0.3% PEI, using a Brandell harvester (Brandell, Gaithersburg, MD). Radioligand binding was quantified by gamma counting of the filters. 2.7. cAMP assays MC3R-transfected COS-1 cells were incubated overnight in serum-free D-MEM with HEPES, l-glutamine and penicillin-streptomycin, and 3.0 ␮Ci/ml of [3 H]-adenine. Cells then were treated with vehicle (D-MEM with HEPES, l-glutamine and penicillin-streptomycin) or MTII for 10 min in the presence of 1 mM isobutylmethylxanthine (IBMX; Sigma–Aldrich, St. Louis, MO). The cells then were rinsed with ice-cold PBS and terminated in 1 ml of 10% tricholroacetic acid (TCA; Fisher Scientific, Pittsburg, PA). The cells then were scraped and centrifuged. The resultant pellet was solublized for 24–48 h in 500 ␮L of 1% SDS in 0.1 M NaOH and assayed for protein content by BCA assay (Pierce, Rockford, IL) according to manufacturer’s instructions. [3 H]cAMP was isolated and measured by passing the supernatant from the centrifuged lysates through sequential dowex and alumina columns before elution into scintillation vials, the radioactivity of which were quantified. Calibration curves of the elution profiles of [3 H]-adenine and [3 H]-cAMP were used to predetermine which fractions of eluant were collected for analysis. All relative measures of [3 H]-cAMP were expressed and analyzed as counts per minute (cpm) per milligram of protein. 2.8. Data analysis The DNA sequences from various PCRs were compared to each other, to the previously published MC3R sequence (accession X70667 [23]), and with the rat genome using internet-based BLAST applications available at the National Center for Biotechnology Information of the National Library of Medicine and National Institutes of Health (www.ncbi.nlm.nih.gov). Open reading frame and amino acid sequence analysis was performed using pDRAW32

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(Version 1.1.74; Acaclone Software; www.acaclone.com). The comparison of multiple amino acid sequences was performed with the aid of MULTALIN [8]. Transmembrane helices were predicted from the present sequence using TopPredII [7]. Concentration–response and radioligand binding analyses were performed using Prism (Version 3.03; GraphPad Software Inc., San Diego, CA) and statistical comparisons were made using SigmaStat (Version 2.03; SPSS Inc., Chicago, IL).

3. Results We used homology-based RT-PCR to generate 16 separate clones from two animals to derive a consensus sequence for the rat MC3R. The resultant consensus sequence contained high levels of homology with a portion of the rat genome on chromosome 3 (within 163,064,900–163,065,900 bp). Our final consensus sequence has been deposited in GenBankTM and given the accession number AY671938 (Fig. 1). Comparison of the predicted amino acid sequences of our cloned MC3R with the previously published rat MC3R (accession X70667 [23]) consistently differed in the nucleotides coding for amino acids in the 78th, 81st and 82nd positions. These individual substitutions occurred within the predicted second transmembrane domain and are shown in Fig. 2. These substitutions were as follows: a cystine in the 78th position and an alanine in the 81st position of our consensus sequence that were each previously described as leucines [23] and an alanine residue in the 82nd position that was previously described as a glutamine [23]. 3.1. Radioligand binding assays Our cloned rat MC3R cDNA was subcloned into an expression vector and transiently transfected into COS1 cells. The binding affinity of the expressed receptors for NDP-MSH then was determined by saturation binding assays. A representative saturation isotherm using [125 I]NDP-MSH is shown in Fig. 3. This analysis revealed high affinity binding for [125 I]-NDP-MSH (KD = 0.52 ± 0.06 nM; Bmax = 1.4 ± 0.1 pmol/mg protein). Analysis of competition curves using ␣-MSH, ␥1 -MSH, NDP-MSH, MTII and SHU9119 revealed similar Ki values to those published previously for NDP-MSH, MTII and SHU9119 (Table 1). The Ki values for ␣-MSH and ␥1 -MSH, however, appeared to be substantially higher than those reported (Table 1). To further investigate these differences, we conducted a two-way ANOVA comparing the Ki values obtained here to those previously published for ␣-MSH [2,22,23,28], ␥1 -MSH [17,22,28] and NDP-MSH [2,23,28]. This analysis revealed significant differences in the binding affinity between the present and previous reports (main effect: F(1,15) = 47.83, p < 0.001) and post hoc tests using the Student Newman–Keuls method highlighted differences between the past and present Ki values for ␣-MSH and ␥1 -MSH, but not

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Fig. 1. Amino acid sequences of the presently described rat MC3R (accession number AY671938) and the mouse (Mus musculus [10]) and human (Homo sapiens [11]) forms of the receptor. Shared residues by at least two of the species are in bold type and asterisks under the sequence are used to indicate residues shared by all three species. The transmembrane domains as predicted from the rat MC3R by TopPredII [7] are indicated.

NDP-MSH. Because we are aware of only two or fewer previous reports of the Ki values for MTII or SHU9119 at the rat MC3R, comparisons of binding by these ligands was not included in the statistical analysis.

Fig. 2. TM2 of the MC3R. The predicted amino acid sequence of TM2 from the spiny dogfish (Squalus acanthias; unpublished, accession #AY560605), zebrafish (Danio rerio [18]), chicken (Gallus gallus [29]), mouse (Mus musculus [10]), rat sequence as described presently (accession #AY671938), pig (Sus scrofa; unpublished, accession number AF451837) and human (Homo sapiens [11]), the sequence as predicted from the nucleotides described in the rat genome [12] and the sequence as described previously [23]. Darkened areas show shared residues.

3.2. Formation of camp MC3R-transfected COS-1 cells demonstrated increased cAMP formation with increasing concentrations of MTII (Fig. 4). Statistical analysis using a one-way ANOVA revealed a significant main effect of MTII on cAMP (F7,23 = 109.2, p < 0.001). Dunnett’s post hoc tests showed that doses of 1 nM or greater produced statistically significant elevations of cAMP when compared to vehicle. The EC50 for the cAMP response to MTII was calculated from three independent experiments to be 2.11 ± 0.35 nM.

4. Discussion The present report describes a nucleotide and predicted amino acid sequence corresponding to a rat MC3R that differs from that previously published [23] by three amino acids. Furthermore, the present sequence shows a higher degree of nucleotide homology with the R. norvegicus genome than

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Table 1 Binding affinities of the rat MC3R in the present and previous reports Present rat MC3R NDP-MSH ␣-MSH ␥1 -MSH MTII SHU9119

0.289 119.85 58.20 9.948

± ± ± ±

0.021 12.89 6.30 1.940

0.639 ± 0.15

Published rat MC3R

References

5.273 ± 2.58 18.12 ± 11.3* 5.51 ± 1.24* 4.77 67.8

[2,23,28] [2,22,23,28] [17,22,28] [2,28]

0.88 1.68

[2,28]

A two-way ANOVA including the present data and that previously published for NDP-MSH, ␣-MSH and ␥1 -MSH revealed a significant difference in binding affinity between the two cloned receptors (main effect of receptor: F(1,15) = 47.83, p < 0.001). Post hoc tests using the Student Newman–Keuls method revealed that Ki values for ␣-MSH and ␥1 -MSH (* indicates p < 0.05), but not NDP-MSH were higher than those in the previous reports. The values are provided as Ki (nM) ± S.E.M. from 3 to 4 independent experiments for the present MC3R clone. The references providing Ki values from previously published analyses are provided. Because fewer than three comparable reports providing the Ki values for MTII and SHU9119 were available, these ligands were not included in the statistical comparisons and the actual values from each report are presented rather than the mean ± S.E.M.

does the previously described sequence and is identical in predicted amino acid sequence. Although the differences in amino acid sequence of the corrected sequence described here may call some question to previous reports using rat MC3R, it should be noted that the strain of rat used in these experiments may account for some, if not all, of the observed differences. Details about the origin of the previously cloned receptor are not provided in the original report [23], nor are they in the description provided in the patent obtained for this receptor (US Patent #6,261,838). Moreover, the GenBankTM record on file with NCBI (X70667 Version GI:396551) contains a number of inconsistencies that make it difficult to interpret any differences between the present construct and that previously reported. First and foremost, the strain and species of rat provided in the GenBankTM submission is listed as the Fisher strain of Rattus rattus, although the Fisher rat is, in fact, a strain of R. norvegicus. Although the information in the submission and the title of the submission indicates that the receptor was cloned from R. rattus, it is highly probable that this receptor was actually cloned from R. norvegicus because R. rattus is rarely used as an experimental animal. An additional inconsistency related to the chromosomal location of the original clone adds to the problematic interpretation of these differences. Specifically, although the present MC3R was found on R. norvegicus chromosome 3, the GenBankTM description of the previously cloned receptor indicates that it was found on chromosome 2. It should be noted that chromosome 3 of R. norvegicus shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20 [12]. Nevertheless, while it remains possible that the differences between the present and the previously reported sequences reflect strain and/or species differences between the receptors, it is impos-

Fig. 3. Radioligand binding of MC3R in transiently transfected COS-1 cells. Panel A shows a representative saturation isotherm of [125 I]-NDP-MSH binding to MC3R. The KD and Bmax were calculated to be 0.52 ± 0.06 nM and 1.4 ± 0.1 pmol/mg protein, respectively, from three independent experiments, each conducted in triplicate. Panel B shows a representative competition experiment using ␣-MSH, ␥1 -MSH, MTII, NDP-MSH and SHU9119. The Ki values calculated from three independent experiments, each performed in triplicate, are provided in Table 1.

Fig. 4. cAMP formation in MC3R-transfected COS-1 cells after treatment with increasing concentrations of MTII. The data are shown as the mean ± S.E.M. from three independent experiments. Based on these data, the EC50 was calculated to be 2.11 ± 0.35 nM.

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sible to make this determination based on the information provided. The differences in amino acid sequence between the present and the previously described receptor occurred in the 78th, 81st and 82nd residues within the predicted second transmembrane domain. The comparison of these residues with MC3R of numerous other species presents what is perhaps the strongest argument against strain/species differences accounting for the altered amino acids. The cystine in the 78th position and alanine in the 81st both are conserved in MC3R of various species, including the spiny dogfish (Squalus acanthias; un-published, accession #AY560605), zebrafish (Danio rerio [18]), chicken (Gallus gallus [29]), mouse (Mus musculus [10]), pig (Sus scrofa; unpublished accession number AF451837) and human (Homo sapiens [11]). A third difference was detected in the alanine residue in the 82nd position, which was previously described as a glutamine [23]. This residue of the consensus sequence is conserved in the zebrafish and mouse MC3R, but occurs as a valine in the spiny dogfish, chicken, pig and human MC3R, but never as a glutamine as described in the previously reported rat MC3R. Radioligand binding and functional data show that the rat MC3R described here binds all of the melanocortin ligands with high affinity and stimulates cAMP formation upon agonist treatment. It is important to note that for three of the five ligands tested, the present binding data are well within the ranges demonstrated for the previously described MC3R. Specifically, the range of KD values for NDP-MSH from three previous reports of radioligand binding using the earlier MC3R was 0.28–3.7 nM [14,17,28], placing the KD calculated here (0.52 ± 0.06 nM) within the range of that observed previously. Furthermore, the Ki values for NDP-MSH were not statistically different from those described previously [2,23,28] suggesting that the three amino acids that differ between these receptors confer little to no alteration in the binding characteristics of NDP-MSH. The present experiments did, however, produce markedly different results with respect to the binding affinities of the endogenous ligands ␣- and ␥1 -MSH, which bound the present MC3R with substantially lower affinity than that described for the previously described MC3R [2,17,22,23,28]. Direct comparison of the two-receptor constructs would be required for the proper interpretation of these differences. Nevertheless, the functional data measuring cAMP formation in response to MTII revealed virtually no difference from previous reports, suggesting that the three differing amino acids in the second transmembrane domain play little or no role in the functional responses of this receptor to the synthetic MTII. The present data provide the first report of the complete, and most likely correct sequence of the rat (R. norvegicus) MC3R. Furthermore, these data demonstrate the incredible utility of the wealth of genomic data that has been made available through the National Center for Biotechnology Information of the National Library of Medicine and National Institutes of Health.

Acknowledgments This work was supported by National Institutes of Health awards DK64012 (D.D.), HL58792 (D.K.Y.) and DK52018 (S.J.F.).

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