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Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor
Regulatory Peptides 99 Ž2001. 1–7 www.elsevier.comrlocaterregrep
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Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor Carrie Haskell-Luevano ) , Eileen K. Monck Department of Medicinal Chemistry, UniÕersity of Florida, GainesÕille, FL 32610-0485, USA Received 2 November 2000; received in revised form 15 November 2000; accepted 15 January 2001
Abstract Agouti-related protein ŽAGRP. is one of two naturally occurring antagonists of G-Protein coupled receptors ŽGPCRs. identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 ŽMC4R. and melanocortin-3 ŽMC3R. receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus andror physiologically functions via a mechanism in addition to competitive antagonism of a-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC 50 values for melanocortin agonists Ž a-MSH, NDP-MSH, and MTII. and a p A 2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Neuropeptide; Feeding; Melanotropin; Obesity; MC4R; Energy Homeostasis
1. Introduction The melanocortin system is a unique G-protein coupled receptor ŽGPCR. pathway that not only includes endogenous agonists and receptors, but also the only two identified naturally occurring antagonists of GPCRs ŽagoutirASP and AGRP. w1,2x, and the single transmembrane spanning mahogany protein, which acts upstream of the melanocortin receptors with the ASP, and possibly AGRP w3,4x ŽFig. 1.. The melanocortin peptides Ž a-, b-, g-melanocyte stimulating hormones and ACTH. are the endogenous agonist ligands for these melanocortin receptors and are derived by post-translational processing of the proopiomelanocortin ŽPOMC. gene transcript. The melanocortin receptor family consists of five isoforms ŽMC1R–MC5R. identified to date w5–11x and stimulates the cAMP second messenger signal transduction pathway.
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Tel.: q1-352-846-2722; fax: q1-352-846-0298. E-mail address: [email protected] ŽC. Haskell-Luevano..
The melanocortin pathway includes five genetic factors that have been linked to energy homeostasis and obesity in mice and humans. The mouse agouti protein ŽASP. w12,13x was first characterized as an antagonist of the skin MC1R and brain MC4R w1x which led to the agouti obesity hypothesis in which the obesity of the agouti ŽAy a . mouse was attributed to chronic antagonism of the MC4R by the agouti protein w1,14,15x. The AGRP protein was demonstrated pharmacologically to competitively antagonize the MC3R and MC4R brain melanocortin receptors w16x, and when ectopically expressed, resulted in an obese phenotype w2,17x. The brain melanocortin receptor ŽMC3R and MC4R. knock out animals have been identified as physiologically participating in the regulation of energy homeostasis w15,18x. Furthermore, a genetic modification of the gene transcripts for POMC Žfrom which the agonist ligands are derived. w19x and MC4R w20–23x in obese humans has been identified. AGRP brain mRNA expression has been identified primarily in the arcuate nucleus of the hypothalamus of the rat and primate w24,25x with neuronal projections to the paraventricular hypothalamic nucleus ŽPVN. w25,26x. Both
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C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
Fig. 1. Summary of the melanocortin system components. The melanocortin agonists consist of the POMC derived ligands that include a- and g-melanocyte stimulating hormones ŽMSH.. The five melanocortin receptors ŽMCXR. identified to date and their respective known physiological functions are indicated. These receptors stimulate the adenylate cyclase second messenger signal transduction pathway to increase intracellular cAMP levels. The only two known naturally occurring antagonists of GPCRs, agouti ŽASP. and the agouti-related protein ŽAGRP. antagonize the melanocortin receptors. Additionally, the single transmembrane spanning mahogany Žmg. protein has been identified as interacting with ASP upstream of the MC1R, and is postulated to interact with AGRP.
the arcuate nucleus and the PVN brain nuclei are well recognized for their participation in energy homeostasis. MC4R protein expression has been identified in the PVN w27x as well as MC3R mRNA expression w7x. AGRP is co-localized in neuropeptide Y ŽNPY. containing neurons w28,29x, and when administered centrally to NPY y ry knockout mice, results in increased food intake w30x. Unexpectedly, the NPY knock out mouse possesses a normal weight phenotype w31x that has subsequently been attributed to a compensatory mechanism involving AGRP w30x. Central administration of AGRP in the MC4R knock out animal resulted in increased food intake w30x, which may be attributed to AGRPs antagonism of the MC3R, or via an unidentified mechanism or target w30x. Twenty-four-hour pretreatment of centrally administered AGRPŽ83–132. followed by administration of the melanocortin agonist MTII w32x, resulted in no statistical difference between the animals that had received saline instead of MTII w33x. These data led the authors of those studies to postulate that AGRP may possess an additional physiological mechanism in addition to competitive antagonism. This study was undertaken to characterize AGRP at a constitutively active MC4R and determine if AGRP possesses inverse agonist activity. Additionally, this study provides experimental evidence that AGRP participates in energy homeostasis via a mechanism in addition to competitive antagonism.
sis. Mutants were prepared by the polymerase chain reaction ŽPCR. using pfu polymerase ŽStratagene. and a complementary set of primers containing the nucleotide mutationŽs. resulting in the desired amino acid residue change. After completion of the PCR reaction Ž958 30 s, 12 cycles of 958 30 s, 558 1 min, 688 9 min. the product was purified ŽQiaquick PCR reaction, Qiagen. and eluted in water. Subsequently, the sample was cut with Dpn1 ŽBiolabs. to lineralize the wildtype template DNA leaving only nicked circularized mutant DNA. This was transformed into competent DH5a E-coli. Single colonies were selected and the presence of the desired mutant was checked by DNA sequencing. The DNA containing the mutant was then excised and subcloned into the HindIIIrXbaI restriction sites of the pCDNA 3 expression vector ŽInvitrogen.. Complete mutant mMC4R sequences were confirmed free of PCR nucleotide base errors by DNA sequencing ŽUniversity of Florida sequencing core facility.. 2.2. Cell culture and transfection Briefly, HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and seeded 1 day prior to transfection at 1 to 2 = 10 6 cellr100-mm dish. Mutant and wildtype DNA in pCDNA 3 expression vector Ž20 mg. were transfected using the calcium phosphate method. Stable receptor populations were generated using G418 selection Ž1 mgrml. for subsequent bioassay analysis. 2.3. b-Galactosidase bioassay
2. Materials and methods 2.1. Receptor mutagenesis Mouse MC4R cDNA Ž1.6-kb fragment. was subcloned into pBluescript ŽStratagene. and was used for mutagene-
HEK-293 cells stably expressing wildtype and mutant receptors were transfected with 4 mg CRErb-galactosidase reporter gene as previously described w34,35x. Briefly, 5000 to 15,000 post transfection cells were plated into 96-well Primera plates ŽFalcon. and incubated
C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
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Table 1 Primary sequences of the agonist and antagonist ligands used in this study
overnight. Forty-eight hours post-transfection the cells were stimulated with 100 ml peptide Ž10y6 –10y12 M. or forskolin Ž10y4 M. control in assay medium ŽDMEM containing 0.1 mgrml BSA and 0.1 mM isobutylmethylxanthine. for 6 h. The assay media was aspirated and 50 ml of lysis buffer Ž250 mM Tris–HCl pH s 8.0 and 0.1% Triton X-100. was added. The plates were stored at y808 overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 ml were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates, 40 ml phosphate-buffered saline with 0.5% BSA was added to each well. Subsequently, 150 ml substrate buffer Ž60 mM sodium phosphate, 1 mM MgCl 2 , 10 mM KCl, 5 mM b-mercaptoethanol, 200 mg ONPG. was added to each well and the plates were incubated at 378. The sample absorbance, OD405 , was measured using a 96 well plate reader ŽMolecular Devices.. The relative protein was determined by adding 200 ml 1:5 dilution Bio Rad G250 protein dye:water to the 10 ml cell lysate sample taken previously, and the OD595 was measured on a 96-well plate reader ŽMolecular Devices.. Data points were normalized both to the relative protein content and non-receptor dependent forskolin stimulation. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments. Data analysis and EC 50 values were determined using nonlinear
regression analysis with the PRISM program Žv2.0, GraphPad.. The antagonistic properties of AGRPŽ83–132. ŽPhoenix Pharmaceuticals. SHU9119 ŽBachem. were determined by the ability of these ligands to competitively displace the MTII agonist ŽBachem. in a dose-dependent manner. The pA 2 values were generated using the Schild analysis method w36x.
3. Results Table 1 summarizes the primary sequences of the agonists and antagonists used in this study. Table 2 summarizes the pharmacology of these melanocortin ligands at the wildtype and constitutively active mMC4Rs. The agonists and synthetic peptide antagonist SHU9119 had nearly identical stimulatory or inhibitory activities at both the wildtype and constitutively active mMC4Rs ŽFig. 2., within experimental error. The synthetic melanocortin antagonist, SHU9119 w37x possess slight partial agonist activity at the constitutively active MC4R, reminiscent of the partial activity observed at the MC3R w37,38x. Fig. 3 compares the competitive antagonistic pharmacological profile of AGRPŽ83–132. at the wildtype receptor and the inverse agonist activity of AGRPŽ83–132. at the constitutively active mMC4R. These data demonstrate that the pharmaco-
Table 2 Summary of the pharmacological b-galactosidase activity in the mMC4 receptors mMC4R receptor
Wild type Constitutively active
Agonist EC 50 ŽnM.
Antagonist value
a-MSH
NDP-MSH
MTH
SHU9119
AGRP Ž87–132.
1.99 " 0.28 0.99 " 0.53
0.18 " 0.05 0.35 " 0.04
0.089 " 0.04 0.071 " 0.04
p A 2 s 10.12 p A 2 s 10.20
p A 2 s 9.39 inverse agonist
The antagonist p A 2 values were determined by using the Schild analysis and the MTII agonist. The standard deviation errors of the mean are indicated. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments.
4 C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7 Fig. 2. Pharmacological properties of the melanocortin agonists MTII, a-MSH, NDP, and synthetic antagonist SHU9119 at the wild-type and constitutively active mouse melanocortin-4 receptors. These receptors were assayed by analyzing their ability to stimulate expression of the cAMP-responsive b-galactosidase reporter gene w33,34x. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments.
C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
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Fig. 3. Comparison of the pharmacological activity of AGRPŽ83–132. at the wildtype and constitutively active melanocortin-4 receptors. At the wildtype receptor, AGRPŽ83–132. is a competitive antagonist with a Schild p A 2 value s 9.39, as previously reported by others Ž2,16.. At the constitutively active melanocortin-4 receptor however, a decrease in b-galactosidase activity is observed, characteristic to decreased levels of activity identified for other compounds characterized as inverse agonist w43x. Thus, the pharmacological activity of AGRPŽ83–132. at the constitutively active melanocortin-4 receptor supports the hypothesis that AGRP may possess inverse agonist activity.
logically active C-terminal of AGRP possesses inverse agonist activity wdecrease in basal b-galactosidase activity in the presence of AGRPŽ83–132.x.
4. Discussion A constitutively active mouse MC4R has been generated and characterized in vitro to possess an increased basal activity Žapproximately 50% the maximal response. in the absence of agonist ligand. The melanocortin ligands a-MSH, NDP-MSH, MTII Žagonists. and SHU9119 Žantagonist. possess pharmacological properties similar to those reported at the wildtype receptor ŽTable 2. w38x. AGRPŽ83–132. however, possesses inverse agonist activity at this constitutively active receptor ŽFig. 3.. These data suggest an alternative physiological mechanism of action for AGRP in addition to competitive antagonism of the brain melanocortin receptors. Additionally, these data provide experimental evidence that AGRP may have a physiological function in and of itself, thus acting both indepen-
dently and in concert with the POMC-derived melanocortin agonist a-MSH. NPY is a potent neuropeptide that stimulates a voracious increase in feeding behavior when administered centrally w39,40x. NPY receptors decrease cAMP levels by coupling with Gi w41x. Both AGRP and NPY are colocalized in neurons originating from the arcuate nucleus of the hypothalamus w28,29x Ža brain nuclei well recognized as important for energy homeostasis and feeding behavior., and the data presented herein suggests that AGRP may also decrease intraneuronal cAMP levels. The physiological role of AGRP as an inverse agonist is supported by the in vivo observations of the AnormalB phenotype of the NPY y ry animals w31x, the feeding behavior observed when AGRP is administered to the MC4Ry ry animals w30x, and the ability of AGRP pretreatment to negate the activity of the agonist MTII w33x. The putative physiological mechanism of AGRP has been to function solely as a competitive antagonist of the POMC derived melanocortin agonists at the brain melanocortin receptors to block increased levels of intra-
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Fig. 4. Illustration of the mechanism of AGRP as ŽA. a competitive antagonist and ŽB. an inverse agonist. The neuronal projection cartoons illustrate that AGRP and NPY are co-localized within the same neurons originating from the arcuate nucleus of the hypothalamus and that the POMC derived agonists are localized to separate neuronal projections w42x. In the cartoon depicted in ŽA., both AGRP and a-MSH must be released simultaneously for AGRP to function as a competitive antagonist at the third neuron containing the melanocortin receptors ŽMCR.. Whereas, in the carton depicted in ŽB., for AGRP to function as an inverse agonist, AGRP is released from a presynaptic neuron with a resulting decrease of cAMP levels. This decrease in cAMP levels would additionally be emphasized by the co-release of NPY to act at the NPY receptors ŽNPY-R..
cellular cAMP and the subsequent signal transduction cascade, Fig. 4A. For this competitive antagonistic mechanism to occur, release from the presynaptic neurons containing POMC-derived agonists and separate presynaptic neurons releasing AGRP w42x must both interact with postsynaptic neurons containing the brain melanocortin receptors ŽMC3R andror MC4R. in concert. A new additional mechanism of action for AGRP can now be postulated, based upon the data presented herein. This hypothesis proposes that AGRP may be released presynapthically to postsynaptic neurons containing melanocortin receptors to decrease basal intracellular cAMP levels ŽFig. 4B.. This mechanism requires only one signal from one neuron for a physiological response at a second neuron, whereas the competitive antagonist mechanism requires two independent signals from two separate neurons, releasing AGRP and POMC derived agonist, respectively w42x occurring nearly simultaneously to generate a physiological response at a third postsynaptic neuron Žexpressing the brain melanocortin receptors.. Thus, this additional mechanism of AGRP action requires only the involvement of the presynaptic AGRP release and is less complicated than the competitive antagonistic mechanism. In conclusion, the data presented in this study demonstrate that one ŽAGRP. of the two only known naturally occurring antagonists of GPCRs identified to date, can result in inverse agonist activity at a constitutively active mouse melanocortin-4 receptor. Additionally, this is the first experimental evidence that AGRP may physiologically participate in the regulation of energy homeostasis via a mechanism independent of competitive antagonism, and importantly, independent of any melanocortin agonist ligands. Finally, to the author’s knowledge, this is
the first report of inverse agonist activity by an endogenously produced neuropeptide in the brain. Note in proof While this manuscript was in proof, a report demonstrating AGRPŽ83–132. is an inverse agonist at the human MC4R was published w44x Acknowledgements This work has been funded by NIH Grant RO1DK57080-01 ŽC.H.L.., the Howard Hughes Medical Institute Research Resources Program, University of Florida and Carrie Haskell-Luevano is a recipient of a Burroughs Wellcome fund Career Award in the Biomedical Sciences. References w1x Lu D, Willard D, Patel IR, Kadwell S, Overton L, Kost T, et al. Agouti protein is an antagonist of the melanocyte-stimulatinghormone receptor. Nature 1994;371:799–802. w2x Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, et al. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 1997;278:135–8. w3x Gunn TM, Miller KA, He L, Hyman RW, Davis RW, Azarani A, et al. The mouse mahogany locus encodes a transmembrane form of human attractin. Nature 1999;398:152–6. w4x Nagle DL, McGrail SH, Vitale J, Woolf EA, Dussault Jr. BJ, DiRocco L, et al. The mahogany protein is a receptor involved in suppression of obesity. Nature 1999;398:148–52. w5x Chhajlani V, Wikberg JES. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett 1992;309:417–20. w6x Mountjoy KG, Robbins LS, Mortrud MT, Cone RD. The cloning of a family of genes that encode the melanocortin receptors. Science 1992;257:1248–51.
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w26x Broberger C, Johansen J, Johansson C, Schalling M, Hokfelt T. The neuropeptide Yragouti gene-related protein ŽAGRP. brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci U S A 1998;95:15043–8. w27x Cowley MA, Pronchuk N, Fan W, Dinulescu DM, Colmers WF, Cone RD. Integration of NPY, AGRP, and melanocortin signals in the hypothalamic paraventricular nucleus: evidence of a cellular basis for the adipostat. Neuron 1999;24:155–63. w28x Hahn TM, Breininger JF, Baskin DG, Schwartz MW. Coexpression of AGRP and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1998;1:271–2. w29x Chen P, Li C, Haskell-Luevano C, Cone RD, Smith MS. Altered expression of agouti related protein ŽAGRP. and its colocalization with neuropeptide and ŽNPY. in the arcuate nucleus of the hypothalamus during lactation. Endocrinology 1999;140:2645–50. w30x Marsh DJ, Miura GI, Yagaloff KA, Schwartz MW, Barsh GS, Palmiter RD. Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues. Brain Res 1999;848:66–77. w31x Erickson JC, Clegg KE, Palmiter RD. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 1996;381:415–21. w32x Al-Obeidi F, Castrucci AM, Hadley ME, Hruby VJ. Potent and prolonged acting cyclic lactam analogues of a-melanotropin: design based on molecular dynamics. J Med Chem 1989;32:2555–61. w33x Hagan MM, Rushing PA, Pritchard LM, Schwartz MW, Strack AM, Van Der Ploeg LH, et al. Long-term orexigenic effects of AgRPŽ83–132. involve mechanisms other than melanocortin receptor blockade. Am J Physiol Regul, Integr Comp Physiol 2000;279:R47– 52. w34x Chen W, Shields TS, Stork PJS, Cone RD. A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal Biochem 1995;226:349–54. w35x Lu D, Vage ¨ DI, Cone RD. A ligand-mimetic model for constitutive activation of the melanocortin-1 receptor. Mol Endocrinol 1998;12:592–604. w36x Schild HO. pA, a new scale for the measurement of drug antagonism. Br J Pharmacol 1947;2:189–206. w37x Hruby VJ, Lu D, Sharma SD, Castrucci AML, Kesterson RA, Al-Obeidi FA, et al. Cyclic lactam a-Melanotropin Analogues of Ac-Nle 4 -cwAsp 5 , DPhe7, Lys10 x-a-MSHŽ4–10.-NH 2 with bulky aromatic amino acids at position 7 show high antagonist potency and selectivity at specific melanocortin receptors. J Med Chem 1995;38: 3454–61. w38x Haskell-Luevano C, Lim S, Yuan W, Cone RD, Hruby VJ. Structure activity studies of the melanocortin antagonist SHU9119 modified at the 6, 7, 8, and 9 positions. Peptides 2000;21:49–57. w39x Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1984;115:427–9. w40x Stanley BG, Leibowitz SF. Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimullant of feeding behavior. Proc Natl Acad Sci U S A 1985;82:3940–3. w41x Gerald C, Walker MW, Criscione L, Gustafson EL, Batzl-Hartmann C, Smith KE, et al. A receptor subtype involved in neuropeptide-Yinduced food intake. Nature 1996;382:168–71. w42x Bagnol D, Lu XY, Kaelin CB, Day HE, Ollmann M, Gantz I, et al. Anatomy of an endogenous antagonist: relationship between Agouti-related protein and proopiomelanocortin in brain. J Neurosci ŽOnline. 1999;19:RC26. w43x Meng F, Wei Q, Hoversten MT, Taylor LP, Akil H. Switching agonist–antagonist properties of opiate alkaloids at the delta opioid receptor using mutations based on the structure of the orphanin FQ receptor. J Biol Chem 2000;275:21939–45. w44x Nijenhuis WAJ, Oosterom J, Adan RAH. AGRPŽ83–132. acts as an inverse agonist on the human-melanocortin-4 receptor. Mol Endocrinol 2001;15:164–71.
Rapid communication
Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor Carrie Haskell-Luevano ) , Eileen K. Monck Department of Medicinal Chemistry, UniÕersity of Florida, GainesÕille, FL 32610-0485, USA Received 2 November 2000; received in revised form 15 November 2000; accepted 15 January 2001
Abstract Agouti-related protein ŽAGRP. is one of two naturally occurring antagonists of G-Protein coupled receptors ŽGPCRs. identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 ŽMC4R. and melanocortin-3 ŽMC3R. receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus andror physiologically functions via a mechanism in addition to competitive antagonism of a-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC 50 values for melanocortin agonists Ž a-MSH, NDP-MSH, and MTII. and a p A 2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Neuropeptide; Feeding; Melanotropin; Obesity; MC4R; Energy Homeostasis
1. Introduction The melanocortin system is a unique G-protein coupled receptor ŽGPCR. pathway that not only includes endogenous agonists and receptors, but also the only two identified naturally occurring antagonists of GPCRs ŽagoutirASP and AGRP. w1,2x, and the single transmembrane spanning mahogany protein, which acts upstream of the melanocortin receptors with the ASP, and possibly AGRP w3,4x ŽFig. 1.. The melanocortin peptides Ž a-, b-, g-melanocyte stimulating hormones and ACTH. are the endogenous agonist ligands for these melanocortin receptors and are derived by post-translational processing of the proopiomelanocortin ŽPOMC. gene transcript. The melanocortin receptor family consists of five isoforms ŽMC1R–MC5R. identified to date w5–11x and stimulates the cAMP second messenger signal transduction pathway.
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Tel.: q1-352-846-2722; fax: q1-352-846-0298. E-mail address: [email protected] ŽC. Haskell-Luevano..
The melanocortin pathway includes five genetic factors that have been linked to energy homeostasis and obesity in mice and humans. The mouse agouti protein ŽASP. w12,13x was first characterized as an antagonist of the skin MC1R and brain MC4R w1x which led to the agouti obesity hypothesis in which the obesity of the agouti ŽAy a . mouse was attributed to chronic antagonism of the MC4R by the agouti protein w1,14,15x. The AGRP protein was demonstrated pharmacologically to competitively antagonize the MC3R and MC4R brain melanocortin receptors w16x, and when ectopically expressed, resulted in an obese phenotype w2,17x. The brain melanocortin receptor ŽMC3R and MC4R. knock out animals have been identified as physiologically participating in the regulation of energy homeostasis w15,18x. Furthermore, a genetic modification of the gene transcripts for POMC Žfrom which the agonist ligands are derived. w19x and MC4R w20–23x in obese humans has been identified. AGRP brain mRNA expression has been identified primarily in the arcuate nucleus of the hypothalamus of the rat and primate w24,25x with neuronal projections to the paraventricular hypothalamic nucleus ŽPVN. w25,26x. Both
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 3 4 - 8
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C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
Fig. 1. Summary of the melanocortin system components. The melanocortin agonists consist of the POMC derived ligands that include a- and g-melanocyte stimulating hormones ŽMSH.. The five melanocortin receptors ŽMCXR. identified to date and their respective known physiological functions are indicated. These receptors stimulate the adenylate cyclase second messenger signal transduction pathway to increase intracellular cAMP levels. The only two known naturally occurring antagonists of GPCRs, agouti ŽASP. and the agouti-related protein ŽAGRP. antagonize the melanocortin receptors. Additionally, the single transmembrane spanning mahogany Žmg. protein has been identified as interacting with ASP upstream of the MC1R, and is postulated to interact with AGRP.
the arcuate nucleus and the PVN brain nuclei are well recognized for their participation in energy homeostasis. MC4R protein expression has been identified in the PVN w27x as well as MC3R mRNA expression w7x. AGRP is co-localized in neuropeptide Y ŽNPY. containing neurons w28,29x, and when administered centrally to NPY y ry knockout mice, results in increased food intake w30x. Unexpectedly, the NPY knock out mouse possesses a normal weight phenotype w31x that has subsequently been attributed to a compensatory mechanism involving AGRP w30x. Central administration of AGRP in the MC4R knock out animal resulted in increased food intake w30x, which may be attributed to AGRPs antagonism of the MC3R, or via an unidentified mechanism or target w30x. Twenty-four-hour pretreatment of centrally administered AGRPŽ83–132. followed by administration of the melanocortin agonist MTII w32x, resulted in no statistical difference between the animals that had received saline instead of MTII w33x. These data led the authors of those studies to postulate that AGRP may possess an additional physiological mechanism in addition to competitive antagonism. This study was undertaken to characterize AGRP at a constitutively active MC4R and determine if AGRP possesses inverse agonist activity. Additionally, this study provides experimental evidence that AGRP participates in energy homeostasis via a mechanism in addition to competitive antagonism.
sis. Mutants were prepared by the polymerase chain reaction ŽPCR. using pfu polymerase ŽStratagene. and a complementary set of primers containing the nucleotide mutationŽs. resulting in the desired amino acid residue change. After completion of the PCR reaction Ž958 30 s, 12 cycles of 958 30 s, 558 1 min, 688 9 min. the product was purified ŽQiaquick PCR reaction, Qiagen. and eluted in water. Subsequently, the sample was cut with Dpn1 ŽBiolabs. to lineralize the wildtype template DNA leaving only nicked circularized mutant DNA. This was transformed into competent DH5a E-coli. Single colonies were selected and the presence of the desired mutant was checked by DNA sequencing. The DNA containing the mutant was then excised and subcloned into the HindIIIrXbaI restriction sites of the pCDNA 3 expression vector ŽInvitrogen.. Complete mutant mMC4R sequences were confirmed free of PCR nucleotide base errors by DNA sequencing ŽUniversity of Florida sequencing core facility.. 2.2. Cell culture and transfection Briefly, HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and seeded 1 day prior to transfection at 1 to 2 = 10 6 cellr100-mm dish. Mutant and wildtype DNA in pCDNA 3 expression vector Ž20 mg. were transfected using the calcium phosphate method. Stable receptor populations were generated using G418 selection Ž1 mgrml. for subsequent bioassay analysis. 2.3. b-Galactosidase bioassay
2. Materials and methods 2.1. Receptor mutagenesis Mouse MC4R cDNA Ž1.6-kb fragment. was subcloned into pBluescript ŽStratagene. and was used for mutagene-
HEK-293 cells stably expressing wildtype and mutant receptors were transfected with 4 mg CRErb-galactosidase reporter gene as previously described w34,35x. Briefly, 5000 to 15,000 post transfection cells were plated into 96-well Primera plates ŽFalcon. and incubated
C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
3
Table 1 Primary sequences of the agonist and antagonist ligands used in this study
overnight. Forty-eight hours post-transfection the cells were stimulated with 100 ml peptide Ž10y6 –10y12 M. or forskolin Ž10y4 M. control in assay medium ŽDMEM containing 0.1 mgrml BSA and 0.1 mM isobutylmethylxanthine. for 6 h. The assay media was aspirated and 50 ml of lysis buffer Ž250 mM Tris–HCl pH s 8.0 and 0.1% Triton X-100. was added. The plates were stored at y808 overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 ml were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates, 40 ml phosphate-buffered saline with 0.5% BSA was added to each well. Subsequently, 150 ml substrate buffer Ž60 mM sodium phosphate, 1 mM MgCl 2 , 10 mM KCl, 5 mM b-mercaptoethanol, 200 mg ONPG. was added to each well and the plates were incubated at 378. The sample absorbance, OD405 , was measured using a 96 well plate reader ŽMolecular Devices.. The relative protein was determined by adding 200 ml 1:5 dilution Bio Rad G250 protein dye:water to the 10 ml cell lysate sample taken previously, and the OD595 was measured on a 96-well plate reader ŽMolecular Devices.. Data points were normalized both to the relative protein content and non-receptor dependent forskolin stimulation. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments. Data analysis and EC 50 values were determined using nonlinear
regression analysis with the PRISM program Žv2.0, GraphPad.. The antagonistic properties of AGRPŽ83–132. ŽPhoenix Pharmaceuticals. SHU9119 ŽBachem. were determined by the ability of these ligands to competitively displace the MTII agonist ŽBachem. in a dose-dependent manner. The pA 2 values were generated using the Schild analysis method w36x.
3. Results Table 1 summarizes the primary sequences of the agonists and antagonists used in this study. Table 2 summarizes the pharmacology of these melanocortin ligands at the wildtype and constitutively active mMC4Rs. The agonists and synthetic peptide antagonist SHU9119 had nearly identical stimulatory or inhibitory activities at both the wildtype and constitutively active mMC4Rs ŽFig. 2., within experimental error. The synthetic melanocortin antagonist, SHU9119 w37x possess slight partial agonist activity at the constitutively active MC4R, reminiscent of the partial activity observed at the MC3R w37,38x. Fig. 3 compares the competitive antagonistic pharmacological profile of AGRPŽ83–132. at the wildtype receptor and the inverse agonist activity of AGRPŽ83–132. at the constitutively active mMC4R. These data demonstrate that the pharmaco-
Table 2 Summary of the pharmacological b-galactosidase activity in the mMC4 receptors mMC4R receptor
Wild type Constitutively active
Agonist EC 50 ŽnM.
Antagonist value
a-MSH
NDP-MSH
MTH
SHU9119
AGRP Ž87–132.
1.99 " 0.28 0.99 " 0.53
0.18 " 0.05 0.35 " 0.04
0.089 " 0.04 0.071 " 0.04
p A 2 s 10.12 p A 2 s 10.20
p A 2 s 9.39 inverse agonist
The antagonist p A 2 values were determined by using the Schild analysis and the MTII agonist. The standard deviation errors of the mean are indicated. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments.
4 C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7 Fig. 2. Pharmacological properties of the melanocortin agonists MTII, a-MSH, NDP, and synthetic antagonist SHU9119 at the wild-type and constitutively active mouse melanocortin-4 receptors. These receptors were assayed by analyzing their ability to stimulate expression of the cAMP-responsive b-galactosidase reporter gene w33,34x. Assays were performed using duplicate or triplicate data points and repeated in at least two independent experiments.
C. Haskell-LueÕano, E.K. Monck r Regulatory Peptides 99 (2001) 1–7
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Fig. 3. Comparison of the pharmacological activity of AGRPŽ83–132. at the wildtype and constitutively active melanocortin-4 receptors. At the wildtype receptor, AGRPŽ83–132. is a competitive antagonist with a Schild p A 2 value s 9.39, as previously reported by others Ž2,16.. At the constitutively active melanocortin-4 receptor however, a decrease in b-galactosidase activity is observed, characteristic to decreased levels of activity identified for other compounds characterized as inverse agonist w43x. Thus, the pharmacological activity of AGRPŽ83–132. at the constitutively active melanocortin-4 receptor supports the hypothesis that AGRP may possess inverse agonist activity.
logically active C-terminal of AGRP possesses inverse agonist activity wdecrease in basal b-galactosidase activity in the presence of AGRPŽ83–132.x.
4. Discussion A constitutively active mouse MC4R has been generated and characterized in vitro to possess an increased basal activity Žapproximately 50% the maximal response. in the absence of agonist ligand. The melanocortin ligands a-MSH, NDP-MSH, MTII Žagonists. and SHU9119 Žantagonist. possess pharmacological properties similar to those reported at the wildtype receptor ŽTable 2. w38x. AGRPŽ83–132. however, possesses inverse agonist activity at this constitutively active receptor ŽFig. 3.. These data suggest an alternative physiological mechanism of action for AGRP in addition to competitive antagonism of the brain melanocortin receptors. Additionally, these data provide experimental evidence that AGRP may have a physiological function in and of itself, thus acting both indepen-
dently and in concert with the POMC-derived melanocortin agonist a-MSH. NPY is a potent neuropeptide that stimulates a voracious increase in feeding behavior when administered centrally w39,40x. NPY receptors decrease cAMP levels by coupling with Gi w41x. Both AGRP and NPY are colocalized in neurons originating from the arcuate nucleus of the hypothalamus w28,29x Ža brain nuclei well recognized as important for energy homeostasis and feeding behavior., and the data presented herein suggests that AGRP may also decrease intraneuronal cAMP levels. The physiological role of AGRP as an inverse agonist is supported by the in vivo observations of the AnormalB phenotype of the NPY y ry animals w31x, the feeding behavior observed when AGRP is administered to the MC4Ry ry animals w30x, and the ability of AGRP pretreatment to negate the activity of the agonist MTII w33x. The putative physiological mechanism of AGRP has been to function solely as a competitive antagonist of the POMC derived melanocortin agonists at the brain melanocortin receptors to block increased levels of intra-
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Fig. 4. Illustration of the mechanism of AGRP as ŽA. a competitive antagonist and ŽB. an inverse agonist. The neuronal projection cartoons illustrate that AGRP and NPY are co-localized within the same neurons originating from the arcuate nucleus of the hypothalamus and that the POMC derived agonists are localized to separate neuronal projections w42x. In the cartoon depicted in ŽA., both AGRP and a-MSH must be released simultaneously for AGRP to function as a competitive antagonist at the third neuron containing the melanocortin receptors ŽMCR.. Whereas, in the carton depicted in ŽB., for AGRP to function as an inverse agonist, AGRP is released from a presynaptic neuron with a resulting decrease of cAMP levels. This decrease in cAMP levels would additionally be emphasized by the co-release of NPY to act at the NPY receptors ŽNPY-R..
cellular cAMP and the subsequent signal transduction cascade, Fig. 4A. For this competitive antagonistic mechanism to occur, release from the presynaptic neurons containing POMC-derived agonists and separate presynaptic neurons releasing AGRP w42x must both interact with postsynaptic neurons containing the brain melanocortin receptors ŽMC3R andror MC4R. in concert. A new additional mechanism of action for AGRP can now be postulated, based upon the data presented herein. This hypothesis proposes that AGRP may be released presynapthically to postsynaptic neurons containing melanocortin receptors to decrease basal intracellular cAMP levels ŽFig. 4B.. This mechanism requires only one signal from one neuron for a physiological response at a second neuron, whereas the competitive antagonist mechanism requires two independent signals from two separate neurons, releasing AGRP and POMC derived agonist, respectively w42x occurring nearly simultaneously to generate a physiological response at a third postsynaptic neuron Žexpressing the brain melanocortin receptors.. Thus, this additional mechanism of AGRP action requires only the involvement of the presynaptic AGRP release and is less complicated than the competitive antagonistic mechanism. In conclusion, the data presented in this study demonstrate that one ŽAGRP. of the two only known naturally occurring antagonists of GPCRs identified to date, can result in inverse agonist activity at a constitutively active mouse melanocortin-4 receptor. Additionally, this is the first experimental evidence that AGRP may physiologically participate in the regulation of energy homeostasis via a mechanism independent of competitive antagonism, and importantly, independent of any melanocortin agonist ligands. Finally, to the author’s knowledge, this is
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