Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP) 83–132 : Interaction between AGRP 83–132 and Melanocortin

Agouti-Related Protein Is Posttranslationally Cleaved byProprotein Convertase 1 to Generate Agouti-RelatedProtein (AGRP)83–132: Interaction between AGRP83–132and Melanocortin Receptors Cannot Be Influenced bySyndecan-3

John W. M. Creemers,* Lynn E. Pritchard,* Amy Gyte, Philippe Le Rouzic, Sandra Meulemans,Sharon L. Wardlaw, Xiaorong Zhu, Donald F. Steiner, Nicola Davies, Duncan Armstrong,Catherine B. Lawrence, Simon M. Luckman, Catherine A. Schmitz, Rick A. Davies, John C. Brennand,and Anne White

Department of Human Genetics (J.W.M.C., S.M.), University of Leuven and Flanders Interuniversity Institute forBiotechnology, B-3000 Leuven, Belgium; School of Medicine (L.E.P., A.G., A.W.) and Faculty of Life Sciences (L.E.P., A.G.,P.L.R., N.D., C.B.L., S.M.L., A.W.), University of Manchester, Manchester M13 9PT, United Kingdom; Department ofMedicine (S.L.W.), Columbia University College of Physicians and Surgeons, New York, New York 10032; Department ofBiochemistry and Molecular Biology (X.Z., D.F.S.), University of Chicago, Chicago, Illinois 60637; and AstraZeneca (D.A.,C.A.S., R.A.D., J.C.B.), Mereside, Cheshire SK10 4TG, United Kingdom

Agouti-related protein (AGRP) plays a key role in energy ho-meostasis. The carboxyl-terminal domain of AGRP acts as anendogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain ofAGRP binds to syndecan-3, thereby modulating the effects ofcarboxyl-terminal AGRP at the MC4-R. This model assumesthat AGRP is secreted as a full-length peptide. In this study wefound that AGRP is processed intracellularly after Arg79-Glu80-Pro81-Arg82. The processing site suggests cleavage byproprotein convertases (PCs). RNA interference and overex-pression experiments showed that PC1/3 is primarily respon-sible for cleavage in vitro, although both PC2 and PC5/6A canalso process AGRP. Dual in situ hybridization demonstratedthat PC1/3 is expressed in AGRP neurons in the rat hypothal-amus. Moreover, hypothalamic extracts from PC1-null mice

contained 3.3-fold more unprocessed full-length AGRP, com-pared with wild-type mice, based on combined HPLC and RIAanalysis, demonstrating that PC1/3 plays a role in AGRP cleav-age in vivo. We also found that AGRP83–132 is more potent anantagonist than full-length AGRP, based on cAMP reporterassays, suggesting that posttranslational cleavage is requiredto potentiate the effect of AGRP at the MC4-R. Because AGRPis cleaved into distinct amino-terminal and carboxyl-terminalpeptides, we tested whether amino-terminal peptides modu-late food intake. However, intracerebroventricular injectionof rat AGRP25–47 and AGRP50–80 had no effect on body weight,food intake, or core body temperature. Because AGRP iscleaved before secretion, syndecan-3 must influence food in-take independently of the MC4-R. (Endocrinology 147:1621–1631, 2006)

THERE IS LITTLE doubt that the melanocortin-4 receptor(MC4-R) plays an important role in coordinating ap-

petite and metabolic rate with perceived metabolic require-ment (reviewed in Ref. 1). In this regard, two sets of neuronsin the hypothalamic arcuate nucleus are particularly impor-tant: a) Proopiomelanocortin (POMC) neurons, which gen-erate endogenous ligands for the MC4-R such as �MSH andACTH (2), and agouti-related peptide (AGRP)/neuropep-tide Y neurons, which generate AGRP, an endogenous MCRantagonist (3, 4). Both sets of neurons are sensitive to a widerange of peripheral signals that indicate metabolic status,

such as leptin, insulin, glucocorticoids, and gut hormones (5,6). AGRP expression is up-regulated in situations of negativeenergy balance (3, 4, 7–9). Also, genetic manipulation ofAGRP expression levels (4, 10, 11) and physiological exper-iments (12–15) demonstrate that AGRP has a potent andlong-term anabolic effect on food intake and metabolic rate.Neuroanatomical data and pharmacological studies supportthe view that AGRP has this effect because it acts as a com-petitive antagonist at the MC4-R (16–19), although alterna-tive mechanisms have been proposed (20–22).

Despite the well-established role of AGRP in regulation ofenergy homeostasis, surprisingly little is known of its post-translational regulation in the hypothalamus. This informa-tion is required to understand fully the physiological role ofAGRP and the mechanism(s) by which it exerts its effects. Todate, most physiological studies of AGRP function in vivohave used a chemically synthesized carboxyl-terminal AGRPfragment, AGRP83–132 (23). Pharmacological studies under-taken in vitro have indicated that this peptide and similarcarboxyl-terminal derivatives, such as AGRP87–132, are suf-

First Published Online December 29, 2005* J.W.M.C. and L.E.P. contributed equally to this work.Abbreviations: AGRP, Agouti-related peptide; ISH, in situ hybrid-

ization; MCR, melanocortin-4 receptor; PC, proprotein convertase; pKb,affinity of the antagonist; POMC, proopiomelanocortin; sh, short hair-pin; shRNAi, shRNA interference; SSC, sodium saline citrate.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/06/$15.00/0 Endocrinology 147(4):1621–1631Printed in U.S.A. Copyright © 2006 by The Endocrine Society

doi: 10.1210/en.2005-1373

1621

ficient to antagonize the MC4-R (16, 18, 24). Moreover, HPLCanalysis of AGRP immunoreactivity in rat hypothalamic ex-tracts indicate that AGRP undergoes posttranslational cleav-age to generate a carboxyl-terminal fragment in vivo and verylittle full-length AGRP remains (25, 26). Likely candidateproteases that may be involved in AGRP processing includeproprotein convertase (PC) 1/3, PC2, and PC5/6a, all ofwhich have a neuroendocrine expression profile and areexpressed in the hypothalamus (27).

However, observations that suggest AGRP is cleaved arenot consistent with the current model of how AGRP andPOMC derived peptides interact at the MC4-R in vivo. It hasbeen proposed that syndecan-3, a central nervous system-specific proteoglycan that is implicated in food intake reg-ulation, acts as a coreceptor for MC4-R by binding to amino-terminal AGRP via its heparin sulfate side chains andpresenting carboxyl-terminal AGRP to the MC4-R (28, 29).This model is consistent with observations in geneticallymanipulated mouse models (28, 30) but implies that AGRPis secreted as a full-length molecule.

Based on these contradictory lines of evidence, it is im-portant to determine whether AGRP undergoes posttrans-lational cleavage because if the syndecan-3 model is correct,then most physiological studies have been based on peptidesthat are not produced in vivo, and more appropriate studiesare needed using full-length AGRP (30). In addition, if car-boxyl-terminal AGRP fragments are produced by posttrans-lational cleavage, then one or more amino-terminal frag-ments must also be produced. These peptides may exertimportant physiological effects that are independent of themelanocortin system. Finally, if AGRP is posttranslationallyprocessed, then the processing pathway may be tightly reg-ulated as a means of controlling the amount of melanocortinantagonist synthesized and secreted at any given time.

In this present study we addressed five questions: 1) isAGRP posttranslationally processed; 2) which PCs (if any)are capable of cleaving AGRP and which AGRP peptides aresecreted; 3) which proprotein convertases are expressed inAGRP neurons; 4) what are the relative potencies of secretedAGRP peptides at the MC4-R; and 5) what are the physio-logical effects of amino-terminal AGRP peptides in rats?

Materials and MethodsGeneration of human AGRP expression constructs

Full-length AGRP, including the signal peptide and a consensusKozak initiation signal, was PCR amplified from a human hypothalamuscDNA library (Clontech Laboratories Inc., Mountain View, CA) usingthe following primers: sense, 5�-AGCCAGGCCATGCTGACCG-CAGCGTTGC-3�, antisense, 5�-CCCAAGCTTCTAGGTGCGGCTG-CAGGGATT-3�. A separate reverse primer was designed to generate aFlag epitope-tagged version of full-length AGRP: 5�- CCCAAGCTTC-TACTTGTCGTCGTCGTCCTTGTAGTCGGTGCGGCTGCAGGGATT-3�. PCR products were directly cloned into pCR-BluntII-TOPO (Invitro-gen, Carlsbad, CA). Inserts were excised with EcoRI and subsequentlycloned into pcDNA3� (Invitrogen). Clones in the correct orientationwere verified by sequence analysis. Subsequently, a series of mutatedclones were generated in which Arg79-X-X-Arg82, Arg85-Arg86, andArg86-X-X-Arg89 were converted to Ala79-X-X-Ala82, Ala85-Ala86, andAla86-X-X-Ala89. Mutagenesis was undertaken using a Quickchangesite-directed mutagenesis kit (Stratagene, La Jolla, CA) following themanufacturer’s protocol. A further two constructs were generated, inwhich internal FLAG epitope tags were incorporated immediately ad-

jacent to the Arg79-X-X-Arg82 and Arg85-Arg86 sites. For the Arg79-X-X-Arg82-FLAG construct, two PCR products were generated usingthe following primer pairs: sense, 5�-AGCCAGGCCATGCTGACCG-CAGCGTTGC-3�, antisense, 5�-CTTGTCGTCGTCGTCCTTGTAGTCG-CGGGGCTCGCGGTCCTG-3� and sense, 5�-GACTACAAGGACGAC-GACGACAAGTCCTCACGTCGCTGCGTA-3�, antisense, 5�-CCCAA-GCTTCTAGGTGCGGCTGCAGGGATT-3�. These two PCR productswere mixed together, diluted, and amplified with the original full-length AGRP primers. The PCR product was cloned into pCR-BluntI-I-TOPO (Invitrogen) and subsequently pcDNA3� (Invitrogen). For theArg85-Arg86-FLAG construct, the following PCR primer pairs wereused: sense, 5�-AGCCAGGCCATGCTGACCGCAGCGTTGC-3�, anti-sense, 5�-CTTGTCGTCGTCGTCCTTGTAGTCGCGACGTGAGGAGC-GGGG-3� and sense, 5�-GACTACAAGGACGACGACGACAAGTGC-GTAAGGCTGCATGAGT-3�, antisense, 5�-CCCAAGCTTCTAGGTGC-GGCTGCAGGGATT-3�.

Transfection of mammalian cells and analysis ofAGRP processing

AtT20 and �TC3 cells were transfected using Lipofectamine (Invitro-gen) and �TC1–6 cells using Lipofectamine 2000 (Invitrogen) as de-scribed previously (31). Regulated secretion experiments were per-formed essentially as described (32), except that secretion was inducedfor 3 h using 60 mm KCl. A truncated soluble form of furin (32) was usedas a control for constitutive secretion. Albumin (25 �g/ml) was addedto the medium samples before precipitation with 4 volumes of methanolat �20 C. Medium precipitates and cells were dissolved in sample bufferand size separated by SDS-PAGE. Western blotting was performed asdescribed (33) using mouse anti-FLAG antibodies M1 or M2 (Sigma-Aldrich, St. Louis, MO) or a rabbit antibody directed against AGRP thatrecognizes both pro-AGRP and AGRP (kindly provided by Dr. G. Barsh,Stanford University School of Medicine, Stanford, CA).

Immunocytochemistry

Indirect immunofluorescence microscopy was performed as de-scribed (34) with some modifications. Briefly, AtT20 cells, fixed in 4%paraformaldehyde, were incubated with mouse anti-FLAG M1 antibodyand a rabbit antibody directed against the amino terminus of POMC(kindly provided by Dr. P. Lowry, University of Reading, Reading, UK)diluted in PBS containing 0.5% blocking reagent (Roche, Indianapolis,IN) and 0.2% Triton X-100. Bound antibodies were detected with fluo-rescently labeled secondary antibodies (Alexa dyes; Molecular ProbesInc., Eugene, OR). Slides were mounted in Vectashield mounting me-dium (Vector Laboratories, Burlingame, CA) and analyzed on a Axio-phot fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany)equipped with UV optics. Images were recorded with a CE200A charge-coupled device camera system (Photometrics Inc., Huntington Beach,CA) using SmartCapture (Digital Scientific, Cambridge, UK) software.

Immunoelectron microscopy

Ultratructural analysis was performed based on the preembeddingimmunolabeling procedure described by Yi et al. (35). Cells were fixedin 3% paraformaldehyde and 0.15% glutaraldehyde. After quenchingwith 0.1% NaBH3, the cells were permeabilized with 0.035% Triton X-100and incubated with primary antibody (1:1000 dilutions of rabbit anti-AGRP polyclonal or mouse anti-ACTH monoclonal). Ultrasmall gold-conjugated secondary antibodies (goat antirabbit or goat antimouse IgG;both Aurion, Wageningen, The Netherlands) were used at 1:100 dilu-tions. After postfixation in 2% glutaraldehyde, silver enhancement wasperformed using Aurion R-Gent SE-EM reagent (Aurion), according tothe guidelines of the supplier. Finally, the cells were osmicated in 0.5%OsO4 and embedded in Agar 100 Resin (Agar Scientific, Essex, UK)Ultrathin sections were cut using the Leica Ultracut UCT ultramicrotomeand stained with uranyl acetate and lead citrate. The sections wereanalyzed on a CM10 transmission electron microscope (Philips, Am-sterdam, The Netherlands).

RNA interference

The 19-mer target sequences for fur and Pcsk6 (PACE4 gene) havebeen described previously (36). The 19-mer target regions of Pcsk1 (PC1/

1622 Endocrinology, April 2006, 147(4):1621–1631 Creemers et al. • AGRP Processing in the Hypothalamus

3), and Pcsk5 (PC5/6A) for RNA interference were selected using smallinterfering RNA (siRNA) Target Finder (Ambion: http://ambion.com/techlib/misc/siRNA_finder.html). The target sequences for Pcsk1 andPcsk5 are 5�-GAAGCGCTCTTCATATCAC-3�, and GACCATTCGAC-CAAACAGT-3�, respectively. Upper and lower 60-mer oligonucleotidesencoding the corresponding short hairpin (sh) RNAs were designedusing pSilencer Converter (Ambion: http://ambion.com/techlib/misc/psilencer_converter.html). shRNAs contain the 19-mer target sequence,a short hairpin loop sequence (TTCAAGAGA) and the antisense targetsequence, flanked by sequences necessary for RNA polymerase III ter-mination (TTTTTT) and cloning. The double-stranded oligonucleotideswere cloned in the mU6pro vector, kindly provided by Dr. D. Turner(37). PC2 was silenced using an engineered �-ribozyme system kindlyprovided by Dr R. Day (38). The efficiency was confirmed by cotrans-fection of 0.8 �g of mU6 pro vector encoding shRNAs or the �-ribozyme,with 0.2 �g expression vectors encoding the target mRNA and 1 �gempty vector.

Generation of recombinant full-length AGRP

Full-length AGRP (minus predicted signal peptide) was expressed inEscherichia coli, purified, and refolded essentially as previously described(17, 39). Briefly AGRP was PCR amplified from a human hypothalamuscDNA library (Clontech) using the primers: sense, 5�-CGGGATCCG-GCTTGGCCCCCAT-3�, antisense, 5�-CCCAAGCTTCTAGGTGCG-GCTGCAGGGATT-3�. The PCR product was digested with BamHI andHindIII and cloned into pT7.36His, an in-house vector that incorporatesa 6-His tag. E. coli BL21 (Invitrogen) were transformed with pT7.36His-AGRP. Expression of the recombinant protein was induced in the pres-ence of 0.4 mmol/liter isopropyl-�-d-thiogalactopyranoside and puri-fied on a Ni-NTA agarose column using the QIAexpress kit (QIAGEN,Crawley, UK) according to manufacturer’s protocols. RecombinantAGRP was refolded following the protocol of Rosenfeld et al. (17). Fiftymicroliters of refolded material were subjected to analytical size-exclu-sion chromatography on a 2.4-ml Superdex 75 column (Amersham Bio-sciences, Chalfont St. Giles, UK) equilibrated in 50 mm Tris-HCl and 0.15m NaCl (pH7.4). The column was eluted with the same buffer at 50�l/min. Protein concentration was determined by both Dc protein assay(Bio-Rad Laboratories, Hercules, CA) and measurement of absorbanceat 280 nm using a ND-1000 spectrophotometer (Nanodrop Technologies,Wilmington, DE), The extinction coefficient for AGRP was calculatedusing the equation of Gill and Von Hippel (40).

Dual in situ hybridization (ISH)

Male Sprague Dawley rats (Charles River Laboratories, Boston, MA)weighing 250–300 g were used. Coronal sections (15 �m) were cutthrough the entire rostrocaudal axis of the rat brain. Sections were thawmounted onto slides, quickly dried, and stored at �80 C. Double-ISHstudies were performed using 33P- plus digoxigenin-labeled ribonucle-otide probes (riboprobes). To generate riboprobes, PCR-amplified cD-NAs encoding rat PC1/3 (accession no. NM-017091, nucleotides 2072–2381), rat PC2 (accession no. NM-012746, nucleotides 1133–1463), and ratneuropeptide Y (accession no. NM-012614, nucleotides 76–426) wereligated into pGem-T (Promega, Madison, WI) using standard protocols.Linearized plasmids were transcribed with either T7 or SP6 accordingto manufacturer’s instructions. Reactions were terminated by digestionof the plasmid template and riboprobes were extracted (33P-labeledriboprobes only), precipitated, and resuspended in 50 �l 50% nuclease-free formamide� 1 �l RNasin (Promega) and stored at 20 C. Riboprobeswere heated to 65 C for 5 min and quenched on ice before addition tothe hybridization buffer.

Before hybridization, slides were quickly brought to room temper-ature and sections were fixed for 15 min in cold 4% paraformaldehydein 0.1 m PBS (pH 7.4). Slides were briefly rinsed in PBS (PB� 0.9% NaCl),acetylated for 10 min in 0.25% acetic anhydride/0.1 m triethanolamine/0.9% NaCl, and then rinsed 3 � 2 min in PBS. Sections were takenthrough an increasing ethanol series, followed by 5 min in chloroform.Air-dried sections were incubated with antisense riboprobes (5 � 105

dpm of 33P-labeled riboprobe/slide plus 30 ng digoxigenin-labeled ri-boprobe/slide) in hybridization buffer [50% deionized formamide, 4�sodium saline citrate (SSC) (pH 7.0), 1 mm EDTA, 20 �g/ml yeast tRNA,10% dextran sulfate, 1� Denhardt’s solution, and 0.25% sodium dodecyl

sulfate], and incubated overnight at 65 C in a moist chamber. Thefollowing day, slides were washed at room temperature for 10 min in2� SSC, followed by 2 � 30-min washes at 60 C, RNase treated [20�g/ml in TEN buffer: 500 mm NaCl, 10 mm Tris (pH 7), and 1 mm EDTA]at 37 C for 30 min and then sequentially washed for 30 min at 60 C in2� SSC/50% formamide, followed by 0.5� SSC.

For the detection of the digoxigenin-labeled riboprobe signal, slides(after high stringency wash) were washed in buffer 1 [100 mm Tris (pH7.5), 150 mm NaCl] for 2 � 10 min and then blocked for 30 min in buffer1 � 0.1% Triton X-100 � 2% heat-inactivated fetal bovine serum (Roche).Antidigoxigenin-alkaline phosphatase conjugated antibody (Invitrogen)was diluted 1:500 in buffer 1 � 0.1% Triton X-100 � 1% fetal bovine serum,and slides were incubated for 1 h in antibody solution at room temperature.Slides were then washed in buffer 1, and incubated for 10 min in buffer 2[0.1 m Tris (pH 9.5), 0.1 m NaCl, 50 mm MgCl2] before color detection.Digoxigenin-labeled probes were visualized by incubating the slides ina chromogen solution containing nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indoyl-phosphate in buffer 2. The color reaction pro-ceeded at room temperature, and once stopped, slides were extensivelywashed (�3 h) in 10 mm Tris (pH 8.0), 1 mm EDTA, and 150 mm NaCl.Sections were briefly dehydrated in 70% ethanol and air dried. Slideswere then dipped in K5 nuclear emulsion (Ilford, Knutsford, UK) forautoradiography.

HPLC/RIA analysis of mouse hypothalamic extracts

Hypothalami from four PC1/3 null and four wild-type mice (41) weredissected using consistent landmarks and were individually homoge-nized in 0.5 ml of 0.1 n HCl, centrifuged at 16,000 � g, and the super-natant from each was analyzed by HPLC as previously described (26).One-milliliter fractions were collected, evaporated in a Speed Vac con-centrator, and dissolved in buffer for AGRP RIA. The column wascalibrated with 5 ng AGRP83–132 (Phoenix Peptides Inc., Belmont, CA)and with 5 ng of full-length AGRP (provided by Dr. G. Barsh, StanfordUniversity School of Medicine, Stanford, CA). AGRP was measured byRIA as previously described (26) with an antiserum raised against hu-man AGRP and directed at the C-terminal end of the molecule, providedby Dr. G. Barsh (25). AGRP83–132 (Phoenix Peptides) was used for thestandard and tracer. Assay sensitivity is 2.5 pg with 50% displacementof tracer at 50 pg.

cAMP reporter assays

cAMP assays were undertaken as previously described (42). Briefly,CHOK1 cells were stably transfected with full-length human MC4-R anda cAMP reporter construct consisting of a cAMP response element andthree vasoactive intestinal peptide enhancer elements upstream of a lacZ reporter gene (kindly provided by Drs. M. Needham and D. Scanlan,AstraZeneca, Cheshire, UK). Cells were grown to complete confluencein DMEM (Sigma), 10% fetal calf serum, 1% HT supplement (Invitrogen),1% nonessential amino acids (Invitrogen), 200 �g/ml G418 (Invitrogen),and 500 �g/ml hygromycin B (Roche). Cells were washed in PBS andharvested. Ligand stocks (2.5 times) were prepared in indicator freeDMEM and 40-�l aliquots were added, in quadruplicate, to poly-lysinecoated 96-well plates. Stocks (10 times) of either full-length AGRP orAGRP83–132 (Phoenix Peptides) were added to appropriate wells in 10-�laliquots. CHOK1 cells expressing the human MC4-R were added eachwell at a density of 50,000 cells/well, and the plate was incubated for 5 hat 37 C/5% CO2. cAMP was detected by addition of 1 mm chlorophenolred-�-d-galactopyranoside (Roche) in buffer containing a final concen-tration of 40 mm Na2HPO4, 40 mm NaH2PO4, 7 mm KCl, and 0.7 mmMgSO4. �-Galactosidase converts chlorophenol red-�-d-galactopyrano-side to give a red color. Results were quantified by reading absorbanceat 590 nm on a Spectrafluor (Tecan, Mannedorf, Switzerland) platereader. Each experiment was performed a minimum of three times withquadruplicate wells. Dose-response data were fitted to a sigmoid curveusing nonlinear squares regression (Origin 6.0, Microcal Software, Inc.,Northampton, MA). Data from dose-response curves were transformedaccording to the method of Arunlakshana and Schild (43) to determinethe affinity of the antagonist (pKb).

Creemers et al. • AGRP Processing in the Hypothalamus Endocrinology, April 2006, 147(4):1621–1631 1623

In vivo analysis of AGRP peptides

All experiments were performed using adult male Sprague Dawleyrats (250–300 g, Charles River Laboratories, Sandwich, UK). Animalswere kept in a 12-h light, 12-h dark cycle at 21 � 1 C with 45 � 10%humidity and free access to food (Beekay International, Hull, UK) andwater. All experiments were performed in accordance with the UnitedKingdom’s Animals (Scientific Procedures) Act (1986). Animals under-went lateral cerebroventricular cannulation (0.8 mm posterior and 1.5mm lateral to bregma and 3.0 mm down from dura) under halothaneanesthesia. After 1 wk of recovery, animals were housed individuallyand left to acclimatize. The free-moving, conscious rats were givenintracerebroventricular injections of 2 nmol AGRP83–132 (Phoenix Pep-tides) and rat equivalent sequences (XM_574228) for human AGRP25–51(rat AGRP25–47 VAPLKGIRRSDQALFPEFSGLSL) and human AGRP54–82(rat AGRP50–80 TAADRAEDVLLQKAEALAEVLDPQNRESRSP) (pep-tides custom synthesized by Bachem, Bubendorf, Switzerland) or vehicle(isotonic sterile saline) in a volume of 2 �l. Immediately after injections, apreweighed amount of food was presented to the animals. Food consump-tion was measured after 1, 2, 4, 8, 24, and 48 h. A temperature-sensitive,precalibrated radiotelemetery transmitter (TA10TA-F40, Data Sciences In-ternational, Minneapolis, MN) was implanted into the peritoneal cavity atthe same time as the ventricular cannulation. The core body temperatureof the animals was measured continuously throughout the 48 h experi-mental period. Correct cannulae placement was verified after the experi-ment by a positive dipsogenic response to a 2-�l icv injection of 100 nghuman angiotensin (Sigma-Aldrich). Only animals that responded wereincluded in the subsequent analysis.

Statistical analysis

Quantitative measurements of AGRP peptides in hypothalamic ex-tracts were compared using a nonparametric Mann-Whitney U test. Inthe food-intake experiments, food intake and body weight gain wereanalyzed using a parametric one-way ANOVA. Core body temperaturewas expressed as change from mean basal values and was analyzed bycalculating area under the curve (C/h) by the trapezoid method. In alltests, P � 0.05 was considered significant.

ResultsAGRP is posttranslationally cleaved in the regulatedsecretory pathway

In the absence of a suitable hypothalamic cell line thatendogenously expresses AGRP, we analyzed AGRP process-ing by transfecting three well-characterized murine neuroen-docrine cell lines, �TC1–6, �TC-3, and AtT20, with pcDNA3(Invitrogen) encoding human full-length AGRP. These cellswere chosen because they represent useful model systems forthe regulated secretory pathway (44), and they endog-enously express PCs that are likely to cleave AGRP in thehypothalamus. �TC1–6 and �TC-3 were derived from mousepancreatic islets and endogenously express PC2 and bothPC1/3 and PC2, respectively. AtT20 cells were derived frommouse anterior pituitary corticotrophs and endogenouslyexpress PC1.

In �TC3 cells transfected with human AGRP (Fig. 1A),Western blot analysis of the cell lysate demonstrates thatfull-length AGRP (12 kDa) is stored intracellularly and un-dergoes posttranslational cleavage to generate a carboxyl-terminal product of approximately 6 kDa. Stimulation of thecells with KCl greatly enhanced secretion of both full-lengthand carboxyl-terminal AGRP into the media, indicating thatAGRP is stored in secretory granules. Figure 1B shows im-munocytochemical evidence that AGRP colocalizes withPOMC-derived peptides in transfected AtT20 cells. Analysisof cells using electron microscopy (Fig. 1B, lower right panel)

demonstrates that AGRP is located in or near large dense-core vesicles. A similar labeling pattern was observed forACTH (data not shown). These observations represent thefirst direct evidence that AGRP is sorted into the regulatorysecretory pathway. Furthermore, it should be noted that thevast majority of AGRP, both intracellular and secreted, is inthe processed 6-kDa form.

AGRP is posttranslationally cleaved after Arg79-X-X-Arg82

Processing of AGRP is likely to involve PCs, a family ofserine proteases that cleave prohormones at basic motifs,usually R/K-R or R-X-X-R (45). Family members includefurin, PC1 (PC3), PC2, PC4, PC5 (PC6), PACE4, and LPC(PC7, PC8), and two more distantly related members, SKI-1and NARC-1 (46, 47). Analysis of the protein sequence ofhuman AGRP demonstrates that there are three potentialprohormone cleavage sites that could potentially liberate acarboxyl-terminal peptide: Arg79-X-X-Arg82, Arg85-Arg86,and Arg86-X-X-Arg89. In an attempt to identify which cleav-age sites are processed in vitro, we generated seven expres-sion constructs in which potential cleavage sites have beendisrupted by site-directed mutagenesis (Fig. 2A). These were

FIG. 1. A, pcDNA3. AGRP transfected into �TC3 cells is predomi-nantly stored intracellularly and is cleaved to generate a carboxyl-terminal 6-kDa fragment. A truncated soluble form of furin (32) isused as control for constitutive secretion and is more predominant inmedia than cell lysates. B, Coimmunofluorescence studies in AtT20cells transfected with pcDNA3. AGRP indicate that AGRP- (red) andPOMC-derived peptides (green) colocalize. Immunoelectron micros-copy (lower right panel) shows silver-enhanced gold particles labelingAGRP predominantly in large dense-core vesicles.


transfected into �TC3 cells, and precipitated media sampleswere analyzed by Western blot (Fig. 2B). In constructs inwhich the Arg79-X-X-Arg82 site was mutated, processing isblocked (constructs 1 and 3). Mutation of the Arg85-Arg86and Arg86-X-X-Arg89 (constructs 2 and 4) did not affectcleavage. These results strongly suggest that AGRP under-goes a posttranslational cleavage event in vitro afterArg79-X-X-Arg82.

The main secreted peptide is AGRP83–132

In parallel to the above experiment, a series of FLAG-tagged constructs were generated to precisely define thesecreted carboxyl-terminal AGRP peptide. Constructs wereengineered so that a FLAG epitope was inserted between theP1 and P1� residues of the putative cleavage sites Arg79-X-X-Arg82 and Arg85-Arg86 (Fig. 2A). A cleavage event wouldtherefore expose a FLAG epitope at the amino-terminus ofthe cleaved peptide. This could be detected by using ananti-FLAG M1 antibody, which detects only the FLAG-tag

with a free amino terminus, whereas the M2 antibody detectsthe FLAG epitope independent of its position in the protein.Figure 2C shows Western blot analysis of media from �TC3cells transfected with the FLAG-tagged constructs. As ex-pected, the M2 anti-FLAG antibody detects both a full-lengthband and a carboxyl-terminal cleaved fragment with all threeconstructs. The cleaved fragment is strongly detected by M1in cells transfected with construct 6. This provides furtherevidence that AGRP is cleaved after Arg79-X-X-Arg82 andAGRP83–132 are secreted. A faint band was also detected incells transfected with construct 5, suggesting some cleavageat Arg85-Arg86.

AGRP is predominantly cleaved by PC1

To assess which propeptide convertases cleave AGRP, wetransfected �TC1–6, AtT20, and �TC3 cells with pcDNA3(Invitrogen) encoding full-length human AGRP. These cellsendogenously express PC2, PC1/3, and PC1/3/PC2, respec-tively. Figure 3A demonstrates that cleavage occurs in allthree of these cell lines, suggesting both PC1 and PC2 cancleave the Arg79-X-X-Arg82. Vectors encoding shRNA in-terference (shRNAi) fragments targeting proprotein conver-tases were then transfected into �TC3 cells. Cotransfectionexperiments with vectors encoding targeted PCs were un-dertaken to assess efficacy of shRNAi silencing (Fig. 3B). Inall cases complete or near complete suppression wasachieved. We found that silencing of PC1/3 in �TC3 cellsresulted in a partial inhibition of AGRP processing, indicat-ing that this enzyme is important in cleavage of AGRP (Fig.3C). Silencing of other PCs in the presence of PC1/3 had noeffect on AGRP processing, indicating that PC1/3 alone issufficient. Figure 3D shows silencing of PC1/3 in AtT20 cells,which almost completely blocked processing, thereby defin-ing a key role for PC1/3 in AGRP cleavage. To assess whichother PCs can cleave AGRP, PC1/3 shRNAi-transfectedAtT20 cells were cotransfected with furin, PACE4, PC5/6A,PC5/6B, and PC2/7B2. This rescue experiment demon-strated that both PC5/6A and PC2 have significant capacityto cleave AGRP in the absence of PC1/3. A potential role forPC2 is further indicated by the observation that AGRP ispartially cleaved when transfected into �TC1–6 cells.

PC1/3 cleaves AGRP in vivo

To determine whether PC1/3 and PC2 colocalize withAGRP in the hypothalamus in vivo, we undertook dual-ISHexperiments. Figure 4 shows representative dark-field auto-radiograms of coronal sections of the rat forebrain. PC1/3generally exhibits a more restricted expression profile thanPC2 but is particularly strongly expressed in the paraven-tricular nucleus and the supraoptic nucleus. PC2 is stronglyexpressed in the hippocampus and the thalamus. The con-sistency of these data with previous studies demonstrates thespecificity of the riboprobes that we have designed (48, 49).High-power bright-field photomicrographs focusing on thearcuate nucleus demonstrate that both PC1/3 and PC2 (silvergrains) are coexpressed in AGRP neurons (dark staining). Wefound that almost all AGRP neurons express both PC1/3 andPC2, thereby implicating a physiological role for these en-zymes in AGRP posttranslational processing.

FIG. 2. A, Full-length AGRP expression constructs. In each case wild-type (W) putative cleavage sites were disrupted by site-directed mu-tagenesis, changing arginines to alanines (M). The position of insertedFLAG epitopes is indicated by black circles. B, Western blot analysisof media samples from �TC3 cells transfected with each construct. C,Western blot analysis of media samples from �TC3 cells transfectedwith FLAG tagged AGRP constructs. M2 antibody detects the FLAGepitope regardless of its position in the protein, whereas M1 detectsonly FLAG epitopes with a free amino-terminus, indicating that thepredominant secreted peptide is AGRP83–132. The position of putativecleavage sites Arg79-X-X-Arg82 (REPR), Arg85-Arg86 (RR), andArg86-X-X-Arg89 (RCVR) are indicated.


Based on experiments in vitro, PC1 appeared to be pre-dominantly responsible for AGRP cleavage. To investigatethe role of PC1 in vivo, we analyzed AGRP processing inhypothalamic lysates from PC1-null mice. Figure 5A showsa representative chromatograph of an individual wild-typeand PC1-null mouse. In wild-type mice, the majority ofAGRP immunoreactivity coeluted with AGRP83–132, indicat-ing that processing occurs in vivo. However, in PC1-nullmice, there was an increase in full-length AGRP immuno-reactivity. Figure 5B shows quantitative analysis of full-length AGRP and AGRP83–132 in four wild-type and four

PC1-null mice. There was no significant difference inAGRP83–132 levels between the two groups. However, therewas significantly more full-length AGRP in the null mice vs.the wild-type mice (648 � 237 vs. 198 � 31 pg/hypothalamus;P � 0.04). The mean percentage full-length AGRP rose from2.7% in wild-type mice to 9.5% in null mice (P � 0.02),indicating that PC1 processes AGRP in vivo, although otherproteases, presumably PC2 and/or PC5/6a, compensate forPC1 in its absence.

AGRP83–132 is more potent than full-length AGRP atthe MC4R

We undertook detailed pharmacological analysis to com-pare the properties of AGRP83–132, the predominant secretedAGRP peptide, with full-length AGRP. Recombinant full-length AGRP was generated and purified from E. coli AGRPwas refolded as previously described (17). Figure 6A showsthe recombinant protein as analyzed by size-exclusion chro-matography. AGRP eluted as a single peak at a retention timeconsistent with a monomeric state, suggesting that the re-combinant AGRP is correctly folded. To test the relativepotencies of full-length AGRP and AGRP83–132 at the MC4-R,cAMP reporter assays were undertaken in CHOK1 cells sta-bly expressed with human MC4-R and a �-galactosidasereporter gene under the control of cAMP response elements.Data were subjected to Schild analysis to determine pKbvalues (Fig. 6, B and C). AGRP 83–132 [pKb � 8.75 � 0.05 (1.8nm)] was significantly more potent as an inhibitor than full-length AGRP [pKb � 7.95 � 0.04 (11 nm)]. Interestingly, atlow concentrations (e.g. 1 nm) full-length AGRP is ineffective,whereas AGRP83–132 significantly antagonizes �MSH. Giventhat concentrations of secreted AGRP in vivo are likely to be

FIG. 3. A, pcDNA3. AGRP transfected into AtT20, �TC1–6, and�TC3 cells, indicating that AGRP is cleaved in each cell line. B, To testshRNAi efficacy, �TC3 cells were cotransfected with 0.2 �g expressionvectors encoding target PC mRNA and either 0.8 mg mU6pro vectorencoding shRNAi (�) or empty vector (�). In each case, complete ornear complete suppression of recombinant target was achieved asassessed by Western blot. C, �TC3 cells cotransfected with vectorsencoding shRNAi particles targeted to PCs. AGRP is cleaved to nearcompletion in cells transfected with shRNAi for furin, PC2, PACE4,and PC5/6A to generate AGRP83–132. PC1/3 shRNAi inhibits process-ing of AGRP by approximately 50%. D, AtT20 cells transfected withshRNAi particles targeted to PC1/3 blocks processing of full-lengthAGRP by approximately 80%. Cotransfection with PC2/7B2 andPC5/6A almost completely rescues cleavage of AGRP.

FIG. 4. Dark-field autoradiograms of coronal sections of rat forebrainincubated with riboprobes for PC1/3 (i, ii) and PC2 (iv, v). PVN,Paraventricular nucleus; TH, thalamus; VMH, ventromedial hypo-thalamus; HC, hippocampus; SON, supraoptic nucleus. Bright-fieldphotomicrographs focusing on the arcuate nucleus demonstrate thatPC1/3 (silver grains) (iii) and PC2 (silver grains) (iv) colocalize withAGRP (dark staining).


low (50), the observed modest differences in pKb valuesbetween full-length and truncated AGRP are likely to bephysiologically significant.

Rat AGRP25–47 and rat AGRP50–80 do not have a role inenergy homeostasis

The observation that AGRP is cleaved raises questionsconcerning the role of the amino-terminal portion of themolecule. In particular, we postulated that interaction ofamino-terminal peptides with syndecan-3 could affect foodintake independently of the melanocortin system. We syn-thesized rat peptides corresponding to the commerciallyavailable human peptides AGRP25–51 and AGRP54–82 anddetermined cumulative food intake in groups of rats injectedwith a single bolus of 2 nmol AGRP83–132, AGRP25–47, andAGRP50–80 (Fig. 7A). As expected, AGRP83–132 had a potenteffect on food intake that was apparent over 48 h, whereasthe amino-terminal peptides did not stimulate food intake.AGRP83–132 also significantly increased body weight, com-pared with the vehicle-treated cohort (Fig. 7B), and de-creased core body temperature (Fig. 7C). This was attribut-

able to a decrease in expression of uncoupling protein-1 inbrown adipose tissue (data not shown). Neither AGRP25–47nor AGRP50–80 had a significant effect on body weight orbody temperature.

FIG. 5. A, A representative chromatograph demonstrating the char-acterization of AGRP immunoreactivity in wild-type and PC1/3-nullmice hypothalami by HPLC. Arrows indicate elution positions of syn-thetic AGRP83–132 and full-length AGRP. B, Quantification of AGRPpeptides in four wild-type (hatched bars) and four PC1/3 null (blackbars) hypothalami. *, P � 0.05.

FIG. 6. A, Recombinant full-length AGRP was generated in E. coli,purified, and refolded as described (17, 39) and subjected to size-exclusion chromatography, which indicates that AGRP is correctlyfolded in a monomeric state. B, CHOK1 cells stably expressinghMC4-R and a �-galactosidase reporter construct were treated withincreasing concentrations of �MSH, and coincubated with 0 nM (E),1 nM (F), 5 nM (�), 10 nM (f), 50 nM (‚), and 100 nM (Œ) of eitherfull-length AGRP or AGRP83–132. Data points represent means ofquadruplicate measurements. Data shown are one representative ofthree independent experiments. For full-length AGRP the curves for0 nM (E) and 1 nM (F) are superimposed.


Discussion

In this study we addressed several important questionsregarding the posttranslational processing and trafficking ofAGRP. First, we provide direct evidence that AGRP is storedintracellularly in secretory granules and is secreted via theregulated pathway. This is consistent with it acting as an

important regulatory neuropeptide. Indeed, analysis ofAGRP content in hypothalamic extracts (7) and secretionexperiments using perifused hypothalamic slices (25, 26) in-dicate that altered secretion of AGRP is more important thansecretion of POMC-derived peptides in eliciting acutechanges in melanocortinergic tone. Therefore, it is importantto understand how AGRP release from secretory granules isregulated and which AGRP derived peptides are produced.

We clearly demonstrate that AGRP is posttranslationallycleaved to produce a carboxyl-terminal fragment. These dataare consistent with previous HPLC analyses of AGRP im-munoreactivity in rat serum and hypothalamic tissue (25, 26).However, the published studies did not precisely define theprimary form of secreted AGRP. By undertaking a series ofsite-directed mutagenesis experiments, we have shown thatcleavage occurs after the Arg79-Glu80-Pro81-Arg82 site to gen-erate AGRP83–132. This observation is important because mostphysiological studies of AGRP function have used AGRP83–132 as it is the main commercially available form (PhoenixPeptides). This was synthesized based on analogy to theprocessing pattern of atrial-natriuretic factor, but there wasno direct evidence that this peptide was produced in vivo (23).Our data support the concept that it is more important toconsider the effects of AGRP83–132 in physiological studiesrather than full-length AGRP (12–14).

By undertaking RNA interference and overexpression ex-periments in a series of neuroendocrine cell lines, we haveshown that AGRP cleavage is predominantly catalyzed byPC1/3, although PC2 and PC5/6A have the capacity tocleave AGRP in the absence of PC1/3 in vitro. However, incontrast to PC1/3, RNA interference silencing of PC2 andPC5/6A did not inhibit AGRP processing in �TC3 cells,indicating that they are not primarily important in the pro-cessing of AGRP. Combined HPLC and RIA analysis of PC1-null mice hypothalami indicate that PC1/3 cleaves AGRP invivo because there is a significant accumulation of unproc-essed full-length AGRP in null vs. wild-type hypothalami.However, it cannot be entirely excluded that processing byother PCs at the same or another cleavage site occurs inhypothalamic neurons in vivo. It is not surprising that geneticablation of PC1/3 results in only a partial reduction of pro-cessing. Similar observations have been made for other neu-ropeptides in the PC1/3 null mice (51) and other PCs (52). Ithas become clear that for many, but not all substrates, alimited redundancy of PCs exists. Here it seems likely thatPC2 and possibly other PCs compensate for the absence ofPC1.

The observation of posttranslational cleavage of AGRP hasimportant implications regarding the mechanism by whichit elicits its physiological effects. It has been demonstratedpreviously that full-length AGRP, but not carboxyl-terminalAGRP, binds to syndecan-1. Based on this observation, it wasproposed that syndecan-3, which unlike syndecan-1 is en-dogenously expressed in the hypothalamus, acts as a core-ceptor for MC4-R (28). Experiments undertaken in syndecan-3-null mice (28–30, 53) and syndecan-1 transgenic mice (28)clearly indicate that syndecan-3 does indeed play an impor-tant role in energy homeostasis. However, our data show thatAGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides before secretion. Therefore, it is difficult to

FIG. 7. A, Mean food intake measurements following intracerebro-ventricular (icv) administration of a single 2-nmol bolus of vehicle (n �6) (f), AGRP83–132 (n � 5) (F), AGRP25–47 (n � 5) (�), and AGRP50–80(n � 6) (Œ). B, Mean body weight changes in rats following icv injectionof vehicle (open bars), AGRP83–132 (diagonal hatched bars), AGRP25–47(horizontal hatched bars), and AGRP50–80 (black bars). C, Core bodytemperatures were measured over a 48-h period in rats injected withvehicle (black lines), AGRP83–132 (light gray lines), AGRP25–47 (dark graylines), and AGRP50–80 (hatched lines). *, P � 0.05; **, P � 0.01; ***, P �0.0001.


envisage how syndecan-3 binding to an amino-terminal frag-ment could influence the effect of the carboxyl-terminal frag-ment. It is theoretically possible that despite intracellularcleavage, AGRP fragments remain associated and form acomplex with syndecan-3, and MC4-R held together by di-sulfide bridges or noncovalent associations. However, wethink this possibility is highly unlikely. First, covalent asso-ciation of AGRP fragments through disulfide bridges can beruled out because no cysteines are present in amino-terminalAGRP. Second, noncovalent association is unlikely becauseimmunoprecipitation of processed AGRP under nondena-turing conditions did not result in coimmunoprecipitation ofthe propeptide (data not shown). These observations indicatethat syndecan-3 cannot act as a coreceptor for the MC4-R.Consequently, syndecan-3 and the carboxyl terminus ofAGRP must act independently in the regulation of food in-take. This would explain the observation that syndecan-3-null mice are resistant to diet-induced obesity (29), whereasAGRP-null mice are not (54). Nevertheless, recent data dosupport the idea that syndecan-3 facilitates the actions ofendogenous MC4-R antagonists because the obese pheno-type observed in agouti lethal yellow mice is attenuated ona syndecan-3-null background (55). This phenomenon must,presumably, be a result of an indirect mechanism, possiblyrelated to the role of syndecan-3 in central nervous systemplasticity (53).

It is possible that amino-terminal AGRP fragments have arole in energy homeostasis independent of the MC4-R, andsuch a role may be mediated by syndecan-3. This possibilityis supported by the observation that syndecan-3 is highlyexpressed in regions of the hypothalamus that receive denseinnervation from AGRP neurons, such as the paraventricularnucleus (19, 28). A recent study implicated amino-terminalAGRP in energy homeostasis. Goto et al. (15) administeredtwo commercially available human carboxyl-terminal AGRPpeptides, AGRP25–51 and AGRP54–82, into rat brains via in-tracerebroventricular cannulae and found that both peptidesincreased body weight. However, these data are difficult tointerpret because amino-terminal AGRP, unlike carboxyl-terminal AGRP, is not particularly well conserved betweenhumans and rats and shows only 66% similarity. In our studywe synthesized the equivalent rat peptides of humanAGRP25–51 and AGRP54–82 and injected them into rat brains.We found that these peptides, in contrast to AGRP83–132, didnot affect body weight, food intake, or core body tempera-ture. Therefore, our results do not support a role for amino-terminal AGRP in the regulation of body weight. However,owing to a lack of amino-terminal AGRP antibodies, it hasnot been possible to ascertain which amino-terminal AGRPpeptides are produced. The commercially available amino-terminal peptides have presumably been synthesized on theassumption that the Lys52-Lys53 site in human AGRP (Lys48-Lys49 in rat) is posttranslationally cleaved, although there isno direct evidence to support this. Further research is there-fore required to determine which amino-terminal AGRPpeptides are produced in vivo and what, if any, functionaleffect they have. Moreover, it has not been ascertained in thisstudy whether amino-terminal fragments of AGRP can ac-tually bind to syndecan-3. This will require further analysis

to determine whether interaction between N-terminal AGRPand syndecan-3 has any physiological significance.

Both in vitro and in vivo studies have demonstrated thatfull-length AGRP displays some bioactivity (4, 17, 18, 39). Inconsidering the implications of AGRP processing, we pre-dicted that full-length pro-AGRP would be less potent as anantagonist than AGRP83–132. In this study we directly com-pared the pharmacological properties of recombinant full-length AGRP and AGRP83–132 in a cAMP reporter assay usingCHO cells stably transfected with MC4-R. Based on Schildanalysis, we demonstrated that full-length AGRP is 6.1-foldless potent than AGRP83–132. This finding is supported byanother recent study that analyzed full-length human AGRPin a reporter gene assay (56). The differences between full-length AGRP and AGRP83–132 could translate into subtledifferences in efficacy in vivo. Indeed, we previously dem-onstrated that subtle changes in POMC-derived peptide po-tency at the MC4-R can lead to profound obesity in vivo (42).

This study is the first to directly address posttranslationalprocessing and trafficking of AGRP. We have found thatAGRP is stored in secretory granules and is cleaved to gen-erate AGRP83–132. Because amino-terminal and carboxyl-ter-minal AGRP are cleaved from one another before secretion,this study strongly suggests that syndecan-3 does not act asa coreceptor for the MC4-R. Further research is thereforerequired to understand the physiological role of syndecan-3.It would be interesting to study how AGRP processing isregulated in the hypothalamus. Our previous studies indi-cate that posttranslational processing of POMC is regulatedin the hypothalamus with respect to energy balance (8).Other studies have shown that hypothalamic expression andactivity of the PCs, PC1/3 and PC2, are altered in variousrodent models of obesity (8, 57–60). Extrapolating from theseobservations, it seems possible that AGRP processing mayalso be regulated as an additional mechanism of controllingmelanocortin tone in the hypothalamus.

Acknowledgments

We thank Mrs. Irene Conwell for technical assistance and Dr. DavidSmith and Dr. Andrew Turnbull of AstraZeneca for advice and helpfuldiscussions.

Received October 28, 2005. Accepted December 20, 2005.Address all correspondence and requests for reprints to: Professor

Anne White, Stopford Building, University of Manchester, Oxford Road,Manchester M13 9PT, United Kingdom. E-mail: [email protected].

This work was supported by the Wellcome Trust, AstraZeneca, andNational Institutes of Health Grant DK57561 (to S.L.W.). A.G. is fundedby a Biotechnology and Biological Sciences Research Council student-ship award.

J.W.M.C., L.E.P., A.G., P.L.R., S.M., S.L.W., X.Z., D.F.S., N.D., C.B.L.,and S.M.L. have nothing to declare. D. A., C.A.S., R.A.D., and J.C.B. areemployed by AstraZeneca. A.W. has received grant support (2003–2005)from AstraZeneca.

References

1. Cone RD 1999 The central melanocortin system and energy homeostasis.Trends Endocrinol Metab 10:211–216

2. Pritchard LE, Turnbull AV, White A 2002 Pro-opiomelanocortin processingin the hypothalamus: impact on melanocortin signalling and obesity. J Endo-crinol 172:411–421

3. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL 1997 Hy-pothalamic expression of ART, a novel gene related to agouti, is upregulatedin obese and diabetic mutant mice. Genes Dev 11:593–602


4. Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, Barsh GS1997 Antagonism of central melanocortin receptors in vitro and in vivo byagouti-related protein. Science 278:135–138

5. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ 2001 Thearcuate nucleus as a conduit for diverse signals relevant to energy homeostasis.Int J Obesity 25:S63–S67

6. Mizuno TM, Makimura H, Mobbs CV 2003 The physiological function of theagouti-related peptide gene: the control of weight and metabolic rate. TrendsMol Med 35:425–433

7. Harrold JA, Williams G, Widdowson PS 1999 Changes in hypothalamicagouti-related protein (AGRP), but not �-MSH or pro-opiomelanocortin con-centrations in dietary-obese and food-restricted rats. Biochem Biophys ResCommun 258:574–577

8. Pritchard LE, Oliver RL, McLoughlin JD, Birtles S, Lawrence CB, TurnbullAV, White A 2003 Pro-opiomelanocortin (POMC) derived peptides in ratcerebrospinal fluid and hypothalamic extracts: evidence that secretion is reg-ulated with respect to energy balance. Endocrinology 144:760–766

9. Mizuno TM, Mobbs CV 1999 Hypothalamic agouti-related protein messengerribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology140:814–817

10. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL 1997 Overexpressionof Agrt leads to obesity in transgenic mice. Nat Genet 17:273–274

11. Makimura H, Mizuno TM, Mastaitis JW, Agami R, Mobbs CV 2002 Reducinghypothalamic AGRP by RNA interference increases metabolic rate and de-creases body weight without influencing food intake. BMC Neurosci 3:18

12. Rossi M, Kim MS, Morgan DGA, Small CJ, Edwards CMB, Sunter D, Abus-nana S, Goldstone AP, Russell SH, Stanley SA, Smith DM, Yagaloff K,Ghatei MA, Bloom SR 1998 A C-terminal fragment of agouti-related proteinincreases feeding and antagonizes the effect of �-melanocyte stimulating hor-mone in vivo. Endocrinology 139:4428–4431

13. Small CJ, Kim MS, Stanley SA, Mitchell JRD, Murphy K, Morgan DGA,Ghatei MA, Bloom SR 2001 Effects of chronic central nervous system ad-ministration of agouti-related protein in pair-fed animals. Diabetes 248–254

14. Hagan MM, Rushing PA, Pritchard LM, Schwartz MW, Strack AM, Van derPloeg LHT, Woods SC, Seeley RJ 2000 Long-term orexigenic effects of AgRP-(83–132) involve mechanisms other than melanocortin receptor blockade. Am JPhysiol 279:R47–R52

15. Goto K, Inui A, Takimoto Y, Yuzuriha H, Asakawa A, Kawamura Y, TsujiH, Takahara Y, Takeyama C, Katsuura G, Kasuga M 2003 Acute intracere-broventricular administration of either carboxyl-terminal or amino-terminalfragments of agouti-related peptide produces a long-term decrease in energyexpenditure in rats. Int J Mol Med 12:379–383

16. Pritchard LE, Armstrong D, Davies N, Oliver RL, Schmitz CA, Brennand JC,Wilkinson GF, White A 2004 Agouti related protein (83–132) is a competitiveantagonist at the human melanocortin-4 receptor: no evidence for differentialinteractions with pro-opiomelanocortin derived ligands. J Endocrinol 180:183–191

17. Rosenfeld RD, Zeni L, Welcher AA, Narhi LO, Hale C, Marasco J, DelaneyJ, Gleason T, Philo JS, Katta V, Hui J, Baumgartner J, Graham M, Stark KL,Karbon W 1998 Biochemical, biophysical and pharmacological characteriza-tion of bacterially expressed human agouti-related protein. Biochemistry 37:16041–16052

18. Yang Y-K, Thompson DA, Dickinson CJ, Wilken J, Barsh GS, Kent SBH,Gantz I 1999 Characterization of agouti-related protein binding to melano-cortin receptors. Mol Endocrinol 13:148–155

19. Bagnol D, Lu X-Y, Kaelin CB, Day HEW, Ollmann M, Gantz I, Akil H, BarshGS, Watson SJ 1999 Anatomy of an endogenous antagonist: relationshipbetween agouti-related protein and proopiomelanocortin in brain. J Neurosci19:RC26 (1–7)

20. Nijenhuis WAJ, Oosterom J, Adan RAH 2001 AgRP (83–132) acts as an inverseagonist on the human melanocortin-4 receptor. Mol Endocrinol 15:164–171

21. Haskell-Luevano C, Monck EK 2001 Agouti-related protein functions as aninverse agonist at a constitutively active brain melanocortin-4 receptor. RegulPeptides 99:1–7

22. Chai B-X, Neubig RR, Millhauser GL, Thompson DA, Jackson PJ, Barsh GS,Dickinson CJ, Li J-Y, Lai Y-M, Gantz I 2003 Inverse agonist activity of agoutiand agouti-related protein. Peptides 24:603–609

23. Quillan JM, Sadee W, Wei ET, Jimenez C, Li J, Chang JK 1998 A synthetichuman agouti-related protein-(83–132)-NH2 fragment is a potent inhibitor ofmelanocortin receptor function. FEBS Lett 428:59–62

24. Jackson PJ, McNulty JC, Yang Y-K, Thompson DA, Chai B, Gantz I, BarshGS, Millhauser GL 2002 Design, pharmacology, and NMR structure of aminimized cystine knot with agouti-related protein activity. Biochemistry41:7565–7572

25. Li J-Y, Finniss S, Yang Y-K, Zeng Q, Qu S-Y, Barsh G, Dickinson C, GantzI 2000 Agouti-related protein-like immunoreactivity: characterization of re-lease from hypothalamic tissue and presence in serum. Endocrinology 141:1942–1950

26. Breen TL, Conwell IM, Wardlaw SL 2005 Effects of fasting, leptin, and insulinon AGRP and POMC peptide release in the hypothalamus. Brain Res 1032:141–148

27. Bergeron F, Leduc R, Day R 2000 Subtilase-like pro-protein convertases: frommolecular specificity to therapeutic applications. J Mol Endocrinol 24:1–22

28. Reizes O, Lincecum J, Wang Z, Goldberger O, Huang L, Kaksonen M, AhimaR, Hinkes MT, Barsh GS, Rauvala H, Bernfield M 2001 Transgenic expressionof syndecan-1 uncovers a physiological control of feeding behaviour by syn-decan-3. Cell 106:105–116

29. Strader AD, Reizes O, Woods SC, Benoit SC, Seeley RJ 2004 Mice lacking thesyndecan-3 gene are resistant to diet-induced obesity. J Clin Invest 114:1354–1360

30. Reizes O, Benoit SC, Strader AD, Clegg DJ, Akunuru S, Seeley RJ 2003Syndecan-3 modulates food intake by interacting with the melanocortin/AGRP pathway. Ann NY Acad Sci 994:66–73

31. Jackson RS, Creemers JWM, Farooqi IS, Raffin-Sanson M-L, Varro A, Dock-ray GJ, Holst JJ, Brubaker PL, Corvol P, Polonsky KS, Ostrega D, Becker KL,Bertagna X, Hutton JC, White A, Dattani MT, Hussain K, Middleton SJ,Nicole TM, Milla PJ, Lindley KL, O’Rahilly S 2003 Small-intestine dysfunc-tion accompanies the complex endocrinopathy of human proprotein conver-tase 1 deficiency. J Clin Invest 112:1550–1560

32. Creemers JW, Usac EF, Bright NA, Van de Loo JW, Jansen E, Van de Ven WJ,Hutton JC 1996 Identification of a transferable sorting domain for the regulatedpathway in the proprotein convertase PC2. J Biol Chem 271:252845–25291

33. Creemers JW, van de Loo JW, Plets E, Hendershot LM, Van de Ven WJ 2000Binding of BiP to the processing enzyme lymphoma proprotein convertaseprevents aggregation, but slows down maturation. J Biol Chem 275:38842–38847

34. van de Loo JW, Teuchert M, Pauli I, Plets E, Van de Ven WJ, Creemers JW2000 Dynamic palmitoylation of lymphoma proprotein convertase prolongs itshalf-life, but is not essential for trans-Golgi network localization. Biochem J352:827–833

35. Yi H, Leunissen J, Shi G, Gutekunst C, Hersch S 2001 A novel procedure forpre-embedding double immunogold-silver labeling at the ultrastructural level.J Histochem Cytochem 49:279–284

36. Jansen S, Stefan C, Creemers JW, Waelkens E, Van Eynde A, Stalmans W,Bollen M, 2005 Proteolytic maturation and activation of autotaxin (NPP2) asecreted metastasis-enhancing lysophospholipase. J Cell Sci 118:3081–3089

37. Yu JY, DeRuiter SL, Turner DL 2002 RNA interference by expression ofshort-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl AcadSci USA 99:6047–6052

38. D’Anjou F, Bergeron LC, Larbi NB, Fournier I, Salzet M, Perreault J-P, DayR 2004 Silencing of SPC2 expression using an engineered � ribozyme in themouse �TC-3 endocrine cell line. J Biol Chem 279:14232–14239

39. Ebihara K, Ogawa Y, Katsuura G, Numata Y, Masuzaki H, Satoh N, TamakiM, Yoshioka T, Hayase M, Matsuoka N, Aizawa-Abe M, Yoshimasa Y,Nakao K 1999 Involvement of agouti-related protein, an endogenous antag-onist of hypothalamic melanocortin receptor, in leptin action. Diabetes 48:2028–2033

40. Gill SC, von Hippel PH 1989 Calculation of protein extinction coefficientsfrom amino acid sequence data. Anal Biochem 182:319–326

41. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, LindbergI, Ugleholdt R, Holst JJ, Steiner DF 2002 Disruption of PC1/3 expression inmice causes dwarfism and multiple neuroendocrine peptide processing de-fects. Proc Natl Acad Sci USA 99:10293–10298

42. Challis BG, Pritchard LE, Creemers JWM, Delplanque J, Keogh JM, Luan J,Wareham NJ, Yeo GSH, Bhattacharyya S, Froguel P, White A, Farooqi IS,O’Rahilly SO 2002 A missense mutation disrupting a dibasic prohormoneprocessing site in pro-opiomelanocortin (POMC) increases susceptibility toearly-onset obesity through a novel molecular mechanism. Hum Mol Genet17:1997–2004

43. Arunlakshana O, Schild HO 1959 Some quantitative uses of drug antagonists.Br J Pharmacol 14:48–58

44. Dey A, Xhu X, Carroll R, Turck CW, Stein J, Steiner DF 2003 Biologicalprocessing of the cocaine and amphetamine-regulated transcript precursors byprohormone convertases, PC2 and PC1/3. J Biol Chem 278:15007–15014

45. Taylor NA, Van de Ven W, Creemers JWM 2003 Curbing activation: Pro-protein convertases in homeostasis and pathology. FASEB J 17:1215–1227

46. Seidah NG, Benjannet S, Wickman L, Marcinkiewicz J, Jasmin SB, StifaniS, Basak A, Prat A, Chretien M 2003 The secretory proprotein convertaseneural apoptosis-regulated convertase 1 (NARC-1): liver regeneration andneuronal differentiation. Proc Natl Acad Sci USA 100:928–933

47. Seidah NG, Prat A 2002 Precursor convertases in the secretory pathway,cytosol and extracellular milieu. Essays Biochem 38:79–94

48. Schafer MKH, Day R 1995 In situ hybridisation techniques to map processingenzymes. Methods Neurosci 23:16–44

49. Schafer MKH, Day R, Cullinan WE, Chretien M, Seidah NG, Watson SJ 1993Gene expression of prohormone and proprotein convertases in the rat CNS: acomparative in situ hybridisation analysis. J Neurosci 13:1258–1279

50. Shen CP, Wu KK, Shearman LP, Camacho R, Tota MR, Fong TM, Van derPloeg LHT 2002 Plasma agouti-related protein level: a possible correlationwith fasted and fed states in humans and rats. J Neuroendocrinol 14:607–610

51. Pan H, Nanno D, Che FY, Zhu X, Salton SR, Steiner DF, Fricker LD, DeviLA, 2005 Neuropeptide processing profile in mice lacking prohormone con-vertase 1. Biochemistry 44:4939–4948


52. Roebroek AJ, Taylor NA, Louagie E, Pauli I, Smeijers L, Snellinx A, LauwersA, Van de Ven WJ, Hartmann D, Creemers JW 2004 Limited redundancy ofthe proprotein convertase furin in mouse liver. J Biol Chem 279:53442–53450

53. Kaksonen M, Pavlov I, Voikar V, Lauri SE, Hienola A, Riekki R, Lakso M,Taira T, Rauvala H 2002 Syndecan 3 deficient mice exhibit enhanced LTP andimpaired hippocampus dependent memory. Mol Cell Neurosci 21:158–172

54. Qian S, Chen H, Weingarth D, Trumbauer ME, Novi DE, Guan X, Yu G, ShenX, Feng Y, Frazier E, Chen A, Camancho RE, Shearman LP, Gopal-Truter S,MacNeil DJ, Van der Ploeg LHT 2002 Neither agouti-related protein norneuropeptide Y is critically required for the regulation of homeostasis in mice.Mol Cell Biol 22:5027–5035

55. Benoit SC, Clegg DJ, Strader AD, Reizes O, Deletion of the syndecan-3 geneattenuates the hyperphagia and obesity of the agouti lethal yellow (Ay) mouse.Program of the 87th Annual Meeting of The Endocrine Society, San Diego, CA,2005, p 545 (Abstract P3-11)

56. de Rijke CE, Jackson PJ, Garner KM, van Rozen RJ, Douglas NR, Kas MJH,Millhauser GL, Adan RAH 2005 Functional analysis of the Ala67Thr poly-

morphism in agouti related protein associated with anorexia nervosa andleanness. Biochem Pharmacol 70:308–316

57. Jing E, Nillni EA, Sanchez VC, Stuart RC, Good DJ 2004 Deletion of the Nhlh2transcription factor decreases the levels of the anorexigenic peptides � mela-nocyte stimulating hormone and thyrotropin-releasing hormone and impli-cates prohormone convertases I and II in obesity. Endocrinology 145:1503–1513

58. Nilaweera KN, Ellis C, Barrett P, Mercer JG, Morgan PL 2003 Precursorprotein convertase 1 gene expression in the mouse hypothalamus: differentialregulation by ob gene mutation, energy deficit and administration of leptin,and coexpression with prepro-orexin. Neuroscience 119:713–720

59. Sanchez VC, Goldstein J, Stuart RC, Hovanesian V, Huo L, Munzberg H,Friedman TC, Bjorbaek C, Nillni EA 2004 Regulation of hypothalamic pro-hormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone. J Clin Invest 114:357–369

60. Berman Y, Mzhavia N, Polonskaia A, Devi LA 2001 Impaired prohormoneconvertases in Cpefat/Cpefat mice. J Biol Chem 276:1466–1473

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.


Documents

Agouti-Related Protein Is Posttranslationally Cleaved by Proprotein Convertase 1 to Generate Agouti-Related Protein (AGRP) 83–132 : Interaction between AGRP 83–132 and Melanocortin