Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide FamilyPeptide Acting through the Calcitonin Receptor-likeReceptor/Receptor Activity-modifying Protein Receptor Complexes*
Received for publication, May 21, 2003, and in revised form, October 15, 2003Published, JBC Papers in Press, November 13, 2003, DOI 10.1074/jbc.M305332200
Jaesook Roh, Chia Lin Chang‡, Alka Bhalla, Cynthia Klein, and Sheau Yu Teddy Hsu§
From the Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University Schoolof Medicine, Stanford, California 94305-5317
Calcitonin, calcitonin gene-related peptide (CGRP),adrenomedullin (ADM), and amylin belong to a uniquegroup of peptide hormones important for homeostasis indiverse tissues. Calcitonin is essential for calcium bal-ance, whereas CGRP and ADM are important for neuro-transmission and cardiovascular and respiratory regu-lation. Based on phylogenetic analysis, we identifiedintermedin as a novel member of the calcitonin/CGRPpeptide family. Analysis of intermedin expression indi-cated that intermedin is expressed primarily in the pi-tuitary and gastrointestinal tract. Intermedin increasedcAMP production in SK-N-MC and L6 cells expressingendogenous CGRP receptors and competed with labeledCGRP for binding to its receptors in these cells. In ad-dition, treatment of 293T cells expressing recombinantcalcitonin receptor-like receptor (CRLR) and one of thethree receptor activity-modifying proteins (RAMPs)showed that a CRLR/RAMP receptor complex is re-quired for intermedin signaling. In contrast to CGRPand ADM, which exhibited a preferential stimulation ofCRLR when co-expressed with RAMP1 and RAMP2 orRAMP3, respectively, intermedin represents a nonselec-tive agonist for the RAMP coreceptors. In vivo studiesdemonstrated that intermedin treatment led to bloodpressure reduction in both normal and spontaneouslyhypertensive rats via interactions with the CRLR/RAMPreceptor complexes. Furthermore, in vivo treatment inmice with intermedin led to suppression of gastric emp-tying activity and food intake. Thus, identification ofintermedin as a novel member of the calcitonin/CGRPpeptide family capable of signaling through CRLR/RAMP receptor complexes provides an additionalplayer in the regulation of peripheral tissues by CRLRand will allow development of new therapeutic agentsfor pathologies associated with diverse vascular andgastrointestinal disorders.
Originally isolated as a polypeptide hormone essential forcalcium balance (1, 2), calcitonin belongs to a group of peptide
hormones including �-CGRP,1 �-CGRP, adrenomedullin(ADM), and amylin (3). Among these tissue-specific peptides,ADM and CGRP are important endocrine and neurocrine inte-grators of homeostasis in the vascular and respiratory systems,whereas amylin is essential for optimal glucose metabolism.The biological actions of these peptides are mediated via bind-ing to two closely related type II G protein-coupled receptors(GPCRs), the calcitonin receptor, and the calcitonin receptor-like receptor (CRLR) (4, 5). Although the calcitonin receptor isthe main mediator for calcitonin action, it also binds amylin.Recent cloning and functional studies have shown that CGRP,ADM, and, to a lesser extent, amylin interact with differentcombinations of CRLR and the three receptor activity-modify-ing proteins (RAMPs) (5, 6). Studies using mutant mice defi-cient in �-CGRP, ADM, or amylin have indicated that CRLRcould be important for cardiovascular morphogenesis, sensoryneurotransmission, inflammatory reactions, nociceptive behav-ior, and glucose homeostasis (7–12). Thus, the physiologicalfunctions of the peptides in this family are determined byreceptor-binding specificity and the tissue expression profilesof individual ligands.
Because the expression of CGRP and its binding sites doesnot overlap in the brain (13), we hypothesized the existence ofadditional calcitonin/CGRP family peptides. Using a phyloge-netic profiling approach to analyze the GenBankTM/EBI DataBank, we identified a novel calcitonin/CGRP family peptide,intermedin, from the genomes of human and other vertebrates.Sequence analysis of the prepropolypeptides of different familygenes indicated that the sequence homology between interme-din and the paralogous peptides is restricted to the maturepeptide and that the intermedin gene evolved during earlyvertebrate evolution. Pharmacological analyses showed thatintermedin signals through CRLR/RAMP receptor complexesand activates the cAMP-dependent pathway in transfectedcells. We show that intermedin signals through the CRLRsignaling system to regulate vascular and gastrointestinalfunctions in vivo.
EXPERIMENTAL PROCEDURES
Cloning, Phylogenetic Analysis, and Expression Profiles of HumanIntermedin—Human intermedin was initially identified from an ex-pressed sequence tag and a genomic sequence (AK024788 andAL096767 in GenBank™), and its identity was verified by PCR ampli-fication using a human Marathon-Ready pituitary cDNA library (Clon-tech). For analysis of intermedin mRNAs in the human digestive sys-
* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF529213.
‡ Supported by a Dean’s fellowship from the Department of Obstet-rics and Gynecology, Chang Gung Memorial Hospital (Tao-Yuan,Taiwan).
§ To whom correspondence should be addressed: Dept. of Obstetricsand Gynecology, Stanford University School of Medicine, A344E, 300Pasteur Dr., Stanford, CA 94305-5317. Tel.: 650-723-7057; Fax: 650-725-7102; E-mail: [email protected].
tem, normalized first strand cDNA preparations were obtained fromClontech. The putative intermedin peptides from fish were deducedbased on a zebrafish expressed sequence tag sequence (AW421384) andpuffer fish genomic sequences (Fugu rubripes; Scaffold_1011). The ratand mouse intermedin sequences were deduced based on expressedsequence tags BQ192607 and BG918210, respectively. Putative pufferfish �-CGRP, �-CGRP, ADM, and amylin sequences were deducedbased on puffer fish sequences Scaffold_9445, Scaffold_6549,JGI_28042, and JGI_8403, respectively.2 The BLOCK MAKER pro-gram3 was used to align the mature peptides from different species.Phylogenetic analysis was carried out using a routine in ClustalW.4 Theconsensus secondary structure of calcitonin/CGRP family peptides waspredicted using the Network Protein Sequence Analysis server.5 ForNorthern blot analysis of intermedin expression, pituitary RNAs wereextracted from pituitary glands obtained from male Sprague-Dawleyrats. Following extraction using TRIzol solution, total RNA was re-solved on formaldehyde-agarose gels and hybridized with a 32P-labeledrat intermedin cDNA probe. The x-ray film was exposed at �80 °C for1 week with intensifying screens.
Peptide Synthesis—Intermedin-related peptides were synthesizedbased on the solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) proto-col and analyzed by reverse-phase high pressure liquid chromatographywith a Vydac C18 analytical column and by mass spectrometry using amatrix-assisted laser desorption ionization time-of-flight Voyager-DERP workstation. Synthetic ADM, �-CGRP, and related peptides wereobtained from Sigma), AnaSpec, Inc. (San Jose, CA), and Bachem (Tor-rance, CA). Radiolabeled 125I-CGRP (2000 Ci/mmol) was from Amer-sham Biosciences. Stocks of different hormones were prepared in dis-tilled water and diluted in culture medium.
Immunoanalysis—Rabbit anti-intermedin antibodies were generatedusing synthetic peptides corresponding to residues 28–47 (MGPAGRQD-SAPVDPSSPHSY) of human intermedin (Strategic Biosolutions,Ramona, CA). This peptide antigen was selected based on the high se-quence identity (85%) found in this region of human and rodent interme-dins and the negligible similarity to other family peptides. The intermedinpeptide was conjugated to keyhole limpet hemocyanin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride before immunization.Antibodies were purified using antigen-conjugated affinity columns. Inimmunoblot analysis, the anti-human intermedin antibody cross-reactedwith synthetic intermedin counterparts from different vertebrates, butnot with paralogous peptides, including calcitonin, CGRP, ADM, andamylin. For immunohistochemical analysis, tissues were obtained fromadult rats, mice, and bullfrogs and analyzed as described (14). To demon-strate that the intermedin transcript encodes the predicted intermedinmature peptide, full-length human intermedin cDNA was subcloned intothe pcDNA3.1 expression vector. For Western blot analysis of intermedinin culture medium, 293T cells were transfected with the intermedinexpression vector using the calcium phosphate precipitation method. For-ty-eight hours after transfection, the serum-free culture medium washarvested and concentrated using a Centricon-3 column. After concentra-tion, the supernatant was boiled for 5 min in denaturing buffer with 100mM dithiothreitol before SDS-PAGE and Western blot analysis usinganti-intermedin antibodies.
Stimulation of cAMP Production in SK-N-MC and L6 Cells by Inter-medin and Related Peptides—Human neuroblastoma SK-N-MC cellsand rat L6 skeletal myoblast cells expressing endogenous CRLR wereobtained from American Type Culture Collection. To estimate adenylylcyclase activation, SK-N-MC and L6 cells (2 � 105 viable cells/well)were plated in 24-well culture dishes in Dulbecco’s modified Eagle’smedium/nutrient mixture F-12 1 day before treatment. Following a 2-hincubation in serum-free Dulbecco’s modified Eagle’s medium/nutrientmixture F-12, cells were treated with testing reagents for 30 min inmedium containing 0.1% bovine serum albumin and 2.5 mM 3-isobutyl-1-methylxanthine (Sigma) to prevent hydrolysis of cAMP by phosphodi-esterases. Following treatment, cells were lysed, and cAMP content wasdetermined by a specific radioimmunoassay (15).
Activation of CRLR/RAMP Receptor Complexes by Intermedin inTransfected 293T Cells—To study the interaction between intermedinand the CRLR/RAMP receptor complexes, we cloned human CRLR,RAMP1, RAMP2, and RAMP3 (accession numbers NP_005786,NP_005846, NP_005845, and O60896, respectively) cDNAs by PCRfrom human Marathon-Ready cDNA libraries using two sets of primers
flanking the full-length coding sequences of each gene. Each cDNA wasverified by DNA sequencing and subcloned into the pcDNA3.1 expres-sion vector. To allow the detection of cell-surface expression of theseproteins, CRLR and RAMPs were tagged at the N terminus of themature protein with a FLAG epitope. Because it has been shown thatepitope tagging affects the RAMP1 protein signaling (16), we used thewild-type RAMP1 construct for analysis of intermedin signaling.HEK293T cells were maintained in 35-mm culture dishes in Dulbecco’smodified Eagle’s medium/Ham’s F-12 medium (Invitrogen) supple-mented with 10% fetal bovine serum, 100 �g/ml penicillin, 100 �g/mlstreptomycin, and 2 mM L-glutamine. The cells were cotransfected with10 �g of CRLR and/or 10 �g of RAMP expression plasmid using thecalcium phosphate precipitation method. Forty-eight hours after trans-fection, cells were washed twice with Dulbecco’s phosphate-bufferedsaline, harvested from culture dishes, and centrifuged at 400 � g for 5min. To determine the level of expression of CRLR and RAMP on thecell surface, the resuspended cells (2 � 106/tube) were incubated withanti-FLAG antibody M1 (50 mg/ml; Sigma) in Tris-buffered saline (pH7.4) containing 5 mg/ml bovine serum albumin and 2 mM CaCl2 (assaybuffer) for 4 h at room temperature in siliconized tubes. Cells were thenwashed twice with 1 ml of assay buffer after centrifugation at 14,000 �g for 15 s. The horseradish peroxidase-conjugated secondary antibody(sheep anti-mouse IgG) was added to the resuspended cell pellets andincubated for 1 h at room temperature. Cells were washed twice with 1ml of assay buffer by repeated centrifugation before determination ofhorseradish peroxidase activity in cell pellets using ECL reagents (Am-ersham Biosciences) and a Lumimark microplate reader (Bio-Rad).Background binding was determined by adding excess amounts of thesynthetic FLAG peptide (Sigma) at a concentration of 100 �g/ml. Forthe assay of adenylyl cyclase activation in transfected cells, cells (2 �105/ml) were placed in 24-well tissue culture plates (Corning Inc.,Corning, NY) and preincubated at 37 °C for 30 min in the presence of2.5 mM 3-isobutyl-1-methylxanthine before hormonal treatment for 4 h.
Receptor Binding Assay—Ligand binding assays were done in sili-conized microcentrifuge tubes at 37 °C for 2 h. Intact SK-N-MC and L6cells were resuspended in binding buffer (20 mM Tris-HCl (pH 7.4), 2mM MgCl2, and 0.1% bovine serum albumin) with 0.06 �g of 125I-CGRPand various concentrations of nonradioactive peptides. After a 2-hincubation at 37 °C, the cell-associated ligand was estimated (15).Radioactivity was determined using a �-counter (EG&G Wallace,Gaithersburg, MD).
Effects of Intermedin on Blood Pressure and Heart Rate in Normaland Hypertensive Rats—Blood pressure measurements were made inconscious male Sprague-Dawley rats and spontaneously hypertensiverats (SHRs; 7–9 weeks of age) pre-adapted to the measurement proce-dure. Indirect systolic pressure was determined by a programmablenon-invasive blood pressure system using the tail-cuff method (Colum-bus Instruments, Columbus, OH). Following attachment of the pres-sure transducer, rats were left undisturbed for 10 min before base-linemeasurements that spanned a 15-min interval. Following base-linemeasurements, rats were injected intraperitoneally with varying dosesof hormones. Blood pressure and heart rate were monitored for 40 minat 20-s intervals. Changes in blood pressure were calculated as theaverage of 30 measurements performed within each 10-min interval.
Effects of Intermedin on Gastric Emptying Activity—Eight-week-oldmale C57BL/6 mice deprived of food for 20 h were given food pellets for90 min before intraperitoneal injection with different hormones orsaline. After treatment, mice were deprived of food again and killed 90min later. The stomach was excised at the pylorus and cardia beforeweighing. Gastric emptying was calculated by comparing the stomachweight of treated mice with the stomach weight of control mice killed atthe time of hormone injection.
Analysis of Ingestive Behavior—Eight-week-old male C57BL/6 micewere housed individually in a regulated environment. Before intraperi-toneal injection with testing reagents, mice were deprived of food for20 h with free access to water. Food intake was measured by placingpre-weighed pellets in the cage and weighing uneaten pellets at 1, 2,and 4 h after treatment.
Statistical Analysis—Differences between treatment groups were an-alyzed by analysis of variance and Student’s t test.
RESULTS
Intermedin as a Calcitonin/CGRP Family Peptide—Wesearched the GenBankTM/EBI Data Bank for sequence motifswith unique primary and secondary structures shared by allcalcitonin/CGRP family peptides using a phylogenetic profilingapproach that has been used to identify novel corticotropin-
2 Available at www.jgi.doe.gov/fugu/index.html.3 Available at blocks.fhcrc.org.4 Available at blocks.fhcrc.org/blocks.5 Available at pbil.ibcp.fr/.
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releasing hormone family peptides (15). Candidate sequenceswere screened for the presence of proteolytic cleavage sitesflanking the putative mature region of the precursor proteins.Based on these criteria, we identified intermedin genes frommammals and teleosts, including zebrafish and a Japanesepuffer fish (Takifugu rubripes). Human intermedin encodes apreproprotein of 148 amino acids with a signal peptide forsecretion at the N terminus (Fig. 1A). Although the overallamino acid sequence of intermedin shows no similarity toknown proteins, a stretch of 47 residues at the C terminus isflanked by dibasic proteolytic cleavage sites at the N terminusand an �-amidation donor residue at the C terminus. Theputative mature region of intermedin shares �28% sequenceidentity with ADM and �20% with CGRP (Fig. 1B). Impor-tantly, the predicted mature region adopts an N-terminal dis-ulfide-bonded loop leading into an �-helix, followed by a disor-dered structure that is shared by all calcitonin/CGRP familypeptides (Fig. 1B). Furthermore, sequence alignment of inter-medin precursors from mammals and teleosts indicated thatsequence conservation in orthologous intermedins is restrictedto the mature region. The mature intermedins of human andfish share �60% similarity, whereas human and rodent inter-medins are 87% identical (Fig. 1B). In addition, the mouse andrat intermedin peptides appear to differ by only one amino acid.Furthermore, analysis of orthologous intermedins indicatedthat the positions of N-terminal dibasic cleavage sites vary bya few amino acids among different species, whereas an arginineresidue seven amino acids downstream of the dibasic cleavagemotif of human intermedin is conserved in all species, suggest-ing that the mature intermedin from human and other speciescould be a 40-amino acid peptide. On the basis of these se-quence analyses, we predicted that a 47-amino acid maturepeptide (intermedin-long (IMDL)) and a shorter 40-amino acidintermedin (intermedin-short (IMDS)) could be generated byproteolytic cleavage at the N-terminal proximate basic residuesfollowed by an amidated C terminus. Because the putativepreproregion of intermedin from diverse vertebrates is not con-served, intermedin is unlikely to encode additional active pep-tides such as the pro-ADM N-terminal 20-amino acid peptidefound in the ADM precursor (17).
Phylogenetic analysis of 12 CGRP family peptides from fishand mammals suggested an ancient evolution for three sub-groups of these peptide hormones, with mammalian and teleostintermedins clustered in a separate branch with ADM andCGRP (Fig. 1C). Thus, intermedin and other family peptidesevolved before the emergence of modern teleosts and tetrapods.Genomic analysis showed that intermedin is located on thedistal arm of human chromosome 22q13 and syntenic mousechromosome 15. In both human and mouse genomes, interme-din neighbors an aldehyde reductase-like gene. In contrast, allother calcitonin/CGRP family genes cluster on human chromo-somes 11 and 12.
Intermedin Activates the cAMP-dependent Pathway in SK-N-MC and L6 Cells via the CGRP Receptor—Pairwise sequencecomparison and phylogenetic tree building based on all GPCRsequences indicated that intermedin is closest to ADM andCGRP, whereas no orphan GPCR shares a close relatednesswith CRLR, the receptor for ADM and CGRP. Thus, CRLR is acandidate receptor for intermedin. To test this hypothesis, wetreated human neuroblastoma SK-N-MC cells and rat L6 skel-etal myoblast cells, known to express different levels of CRLRand RAMPs (18), with synthetic intermedin peptides and thenmonitored cAMP production. As shown in Fig. 2 (A and B),treatment with the amidated IMDL (amino acids 1–47) orIMDS (amino acids 8–47) peptide resulted in dose-dependentincreases in cAMP production in both cell lines. The observed
activation is specific, as treatment with a nonamidated form ofintermedin, a truncated amidated intermedin fragment (inter-medin (IMD)-(17–47)), or a 31-amino acid peptide from thepreproregion of human intermedin (prointermedin-(55–85))had no effect in either cell line (data not shown), suggestingthat �-amidation and residues 8–16 of intermedin are impor-tant for intermedin bioactivity. Consistent with earlier reports(5, 18, 19), both ADM and �-CGRP also stimulated cAMPproduction in these cell lines (Fig. 2, A and B). Of importance,the stimulatory effect of intermedin was suppressed by co-treatment with a CGRP receptor antagonist (CGRP-(8–37)) inL6 cells (Fig. 2C), demonstrating that intermedin activates thecAMP-dependent pathway via the CGRP receptor (20). To fur-ther characterize the specific action of intermedin on cAMPproduction, L6 cells were co-treated with a putative intermedinC-terminal receptor-binding domain (IMD-(17–47)) or an anti-intermedin polyclonal antibody. As shown in Fig. 2D, IMD-(17–47) was found to be a functional antagonist of intermedinaction, consistent with the observed antagonistic effect of N-terminally truncated CGRP-(8–37) (Fig. 2C) (20). In addition,co-treatment with the anti-intermedin antibody blocked thestimulatory effect of intermedin, whereas co-treatment with anantibody raised against the unrelated stresscopin-related pep-tide (SRP)/urocortin II had no effect (Fig. 2D).
To establish a direct interaction between intermedin andCRLR, we used iodinated CGRP as the radioligand for receptorbinding assays. As shown in Fig. 2 (E and F), IMDL and IMDSdisplaced 125I-CGRP binding to the SK-N-MC and L6 cellsdose-dependently.
Intermedin Is a Nonselective Agonist for CRLR/RAMP Recep-tor Complexes—CGRP and adrenomedullin mediate their ac-tion through the CRLR/RAMP complexes, consisting of CRLRand one of the three RAMP polypeptides. To investigate therole of CRLR/RAMP receptor complexes in intermedin signal-ing, we treated 293T cells expressing different combinations ofrecombinant CRLR and/or RAMPs with intermedin and relatedpeptides. As shown in Fig. 3A, treatment of intermedin, CGRP,or ADM had no effect on cAMP production in 293T cells ex-pressing CRLR alone, whereas calcitonin increased cAMP pro-duction dose-dependently via the endogenous calcitonin recep-tor. In contrast, intermedin dose-dependently stimulatedcAMP production in cells expressing different CRLR/RAMPreceptor complexes (Fig. 3, B–D). Consistent with earlier stud-ies, CGRP and ADM exhibited a preferential stimulation ofCRLR when co-expressed with RAMP1 and RAMP2 or RAMP3,respectively. Compared with CGRP, intermedin exhibited agreater potency in the stimulation of cAMP production in cellsexpressing CRLR/RAMP3, but had lower activity with CRLR/RAMP1. In contrast, intermedin had a lower potency in theactivation of both CRLR/RAMP2 and CRLR/RAMP3 comparedwith adrenomedullin. Thus, the overall rank of potency for thestimulation of CRLR/RAMP1, CRLR/RAMP2, and CRLR/RAMP3 is CGRP � IMD � ADM, ADM � IMD � CGRP, andADM � IMD � CGRP, respectively. Furthermore, consistentwith earlier reports, the expression of CRLR/RAMP receptorcomplexes on the cell surface of transfected cells was found tobe increased synergistically by co-expressing CRLR andRAMPs (Fig. 3E).
Intermedin Expression in the Pituitary and Stomach—Initialreverse transcription-PCR analysis showed that the interme-din transcript is expressed in the pituitary and stomach.Northern blot analysis of rat pituitary RNA showed that twospecific intermedin transcripts of �5 and 2.5 kb were presentin the pituitary (Fig. 4A). To further characterize the expres-sion profile of intermedin, four independent antibodies weredeveloped using a C-terminal 20-amino acid intermedin pep-
Intermedin Is a Novel Calcitonin/CGRP Family Peptide7266
tide (IMD-(28–47)). As shown in Fig. 4B, the anti-intermedinantibody (C2411-2) is specific for intermedin and showed nocross-reaction with related peptides, including calcitonin,CGRP, ADM, and amylin. Using the specific anti-intermedin
antibody, immunohistochemical analysis of �20 differentmouse tissues confirmed intermedin expression in the pituitaryand stomach. As shown in Fig. 4 (C (magnification �100) andD (magnification �200)), intermedin was expressed mainly in
FIG. 1. Cloning of intermedin andelucidation of its identity. A, the hu-man intermedin gene encodes a 148-a-mino acid open reading frame with a 24-amino acid signal peptide for secretion atthe N terminus. Amino acid positions areon the right, and the stop codon is markedwith an asterisk. The putative maturepeptide is underlined, and the N-terminalsignal peptide for secretion is lightlyshaded. The ATG start site and the puta-tive C-terminal amidation donor residueare in boldface. The putative basic cleav-age sites are darkly shaded. B, compari-son of CGRP-related peptides (�-CGRP,�-CGRP, amylin, ADM, and IMD) frommammals and fish. Sequence alignmentof the precursors of these peptides fromhuman indicated that the sequence ho-mology between intermedin and paralo-gous peptides is restricted to the maturepeptide, and no similarity was found inthe putative preproregion. The two inter-medins from puffer fish (T. rubripes) areindicated as IMD1 and IMD2, respec-tively. The putative secondary structuresof mature CGRP peptides are indicatedabove the alignment, and the putativesecondary structures of mature interme-dins are shown below the alignment. Inaddition, the predicted IMDL and IMDSpeptides are indicated by dotted lines un-derneath the alignment. Curved lines,random coil; round cylinder, extendedstrand; wavy banners, helix. The two cys-teines and one neighboring threonine res-idue shared by all aligned peptides areboxed. Residues shared by ADM and in-termedin from different vertebrates areindicated by asterisks. Residues shared byhighly conserved orthologous sequencesof each gene are lightly shaded. The N-terminal arginine residue found in all in-termedins from different species is shownin boldface. h, human; m, mouse; p, pufferfish; r, rat; z, zebrafish. C, the phyloge-netic relationship among 12 representa-tive CGRP-related peptides.
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the intermediate lobe of the pituitary, with sporadic signals inthe anterior lobe. In contrast, negative controls using preim-mune serum or anti-intermedin antibodies presaturated withthe intermedin antigen showed no specific signals (Fig. 4, Eand F). Likewise, immunohistochemical analysis of pituitarysections from rats and bullfrogs showed that intermedin ex-pression was restricted to the intermediate and anterior lobesof the pituitary (Fig. 4, G and H). Because melanin-stimulatinghormone (MSH) had a similar expression pattern in the pitui-tary (Fig. 4I, anti-MSH staining), we tested whether the anti-intermedin antibody cross-reacts with the MSH peptide. As
shown in Fig. 4J, the specific staining of intermedin in thepituitary was not abolished by preincubation with an MSHpeptide. To further demonstrate that the intermedin mRNAencodes the predicted mature intermedin peptide, a humanintermedin cDNA was subcloned in the eukaryotic expressionvector pcDNA3.1, and the expression of intermedin peptidefrom this construct was investigated using transfected 293Tcells. Western blot analysis of concentrated culture mediashowed that cells transfected with the intermedin expressionvector secreted an �5-kDa mature intermedin peptide into theculture medium, whereas the culture medium from cells trans-
FIG. 2. Intermedin shares receptors with CGRP and ADM. A and B, synthetic intermedin peptides (IMDL and IMDS) stimulate cAMPproduction in human neuroblastoma SK-N-MC cells and rat L6 skeletal myoblast cells, respectively. No stimulation by a nonamidated form ofintermedin, a truncated amidated intermedin fragment (IMD-(17–47), or a 31-amino acid peptide from the preproregion of intermedin(prointermedin-(55–85)) was observed (data not shown). Data are means � S.E. (n � 4). C, blockage of the stimulatory effect of intermedin oncAMP production by a CGRP receptor antagonist (CGRP-(8–37)) in L6 cells. D, blockage of the stimulatory effect of intermedin by IMD-(17–47)(1 �M) and the anti-intermedin antibody (anti-IMD Ab) in L6 cells. Data are means � S.E. (n � 4). No effect was observed upon co-treatment withan anti-SRP/urocortin II antibody (anti-SRP Ab). *, significantly different from controls (p � 0.05). E and F, competitive displacement by unlabeledintermedin and related peptides of 125I-CGRP bound to SK-N-MC and L6 cells, respectively. Data are means � S.E. (n � 3).
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fected with the empty vector displayed no signal (Fig. 4K).To characterize the expression of intermedin in the gastro-
intestinal tract, a panel of human cDNAs from the gastrointes-tinal tract was analyzed by PCR. As shown in Fig. 5A (firstpanel, 1 ng of cDNA template/tube), the expression of theintermedin transcript could be detected in the esophagus,stomach, jejunum, ileum, ileocecum, ascending colon, trans-verse colon, descending colon, and rectum. PCR analysis usinga lower amount of cDNA templates (10 pg/tube) showed thatthe expression of the intermedin transcript was greater in the
stomach and jejunum (Fig. 5A, second panel). Furthermore,immunohistochemical staining showed that intermedin wasfound primarily in the muscularis mucosae layer of the stom-ach (Fig. 5B) and that the signal was abolished by presatura-tion with the intermedin antigen (Fig. 5C).
Systemic Hypotensive Action of Intermedin—Because the re-lated ADM is one of the most potent vasodilators (5) and thepituitary-derived intermedin could be released into the sys-temic circulation to act on diverse peripheral tissues, we testedthe effect of intermedin on blood pressure regulation in normal
FIG. 3. Intermedin activates recombinant CRLR/RAMP receptor complexes in transiently transfected 293T cells. Treatment of 293Tcells transiently transfected with an empty expression vector or a CRLR expression vector with intermedin, CGRP, or ADM had no effect on wholecell cAMP production (A). In contrast, calcitonin increased cAMP production dose-dependently through the endogenous calcitonin receptor. Unlikecells expressing CRLR solely, treatment with intermedin increased cAMP production in cells expressing CRLR with RAMP1 (B), RAMP2 (C), orRAMP3 (D). Likewise, treatment with CGRP or ADM stimulated cAMP production in cells expressing CRLR/RAMP receptor complexes withdifferent potency (B–D; n � 3). Shown are the results of indirect binding analysis of cell-surface expression of CRLR and RAMPs using horseradishperoxidase-conjugated sheep anti-mouse antibodies and anti-FLAG epitope antibodies (E). Expression of FLAG epitope-tagged CRLR and RAMPson the cell surface of transfected cells was increased by co-transfection with CRLR and RAMP expression vectors compared with transfection witha single expression vector encoding CRLR or RAMP.
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rats and SHRs using a noninvasive monitoring approach. Asshown in Fig. 6A, intraperitoneal administration of IMDL orIMDS dose-dependently suppressed blood pressure in normal
Sprague-Dawley rats, similar to that induced by ADM. In ad-dition, treatment of IMDL or IMDS also increased heart rate,as found for ADM (Fig. 6B). In contrast, administration of the
FIG. 4. Expression of intermedin inthe pituitary. A, Northern blot analysisshowing that two specific intermedintranscripts were expressed in rat pitui-tary cells. The positions of 28 S and 18 SRNAs are indicated. B, Western blot anal-ysis of synthetic peptides using an anti-intermedin antibody generated againstthe C-terminal 20 amino acids of humanintermedin (MGPAGRQDSAPVDPSSP-HSY). The anti-intermedin antibody isspecific for intermedin and showed nocross-reaction with CGRP, calcitonin,ADM, or amylin. Molecular mass markersare shown on the left, and specific bandsare indicated by arrows. C–F, immunohis-tochemical staining of mouse pituitarysections using the anti-intermedin anti-body (C, magnification �100; D, magnifi-cation �200), preimmune rabbit serum(E), or anti-intermedin antibody presatu-rated with the intermedin ligand (F). Sec-tions incubated with preimmune serum(E) or antibody presaturated with the in-termedin peptide antigen (F) showednegligible signals. G and H, immunohis-tochemical analysis of intermedin expres-sion in pituitary sections from rat andbullfrog, respectively. I and J, immuno-histochemical staining of mouse pituitarysections using an anti-MSH antibody orthe anti-intermedin antibody presatu-rated with an MSH peptide, respectively.Specific signals are indicated by arrows.AL, anterior lobe; IL, intermediate lobe;PL, posterior lobe. K, Western blot anal-ysis of concentrated culture medium from293T cells transfected with an intermedinexpression vector. The anti-intermedinantibody detected an �5-kDa mature in-termedin peptide in the culture medium,whereas the culture medium from cellstransfected with the empty vector dis-played no signal. Specific intermedin sig-nals are indicated by the arrow. Positivesignals from the synthetic intermedinpeptide are shown in the left lanes.
Intermedin Is a Novel Calcitonin/CGRP Family Peptide7270
truncated IMD-(17–47) fragment (Fig. 6C) or the prointermedin-(55–85) peptide (data not shown) had no effect on blood pres-sure regulation. Because intermedin signals through CRLR/RAMP receptor complexes, the ability of a CGRP receptorantagonist (CGRP-(8–37)) to block the actions of intermedinwas also studied. As shown in Fig. 6C, treatment with a 20-foldexcess of CGRP-(8–37) significantly decreased the hypotensiveeffects of IMDL. Likewise, co-treatment with the putative in-termedin receptor-binding domain fragment IMD-(17–47)blocked the hypotensive effects of IMDL. In addition, we stud-ied the hypotensive effect of intermedin in SHRs. IMDL treat-ment reduced blood pressure in SHRs (similar to the effect innormal rats), and the hypotensive effects of IMDL were abol-ished by co-treatment with CGRP-(8–37) (Fig. 6D). In contrast,co-treatment with the low affinity ADM-(22–52) fragment hada minimal effect (21). Thus, intermedin is a specific ligand forthe vascular CRLR/RAMP signaling system and could be im-portant in the mediation of vascular responses for homeostasis.
Intermedin Suppresses Food Intake and Gastric Emptying—Earlier studies have shown that both CGRP and ADM havepotent anorexic effects (22) and could mediate actions throughcentral or peripheral CRLR/RAMP systems. To examinewhether intermedin has a role in anorexia regulation, we stud-ied the ability of intermedin to regulate feeding behavior basedon cumulative food intake in fasted mice. Intraperitoneal in-jection with IMDL, IMDS, ADM, or a type II corticotropin-releasing hormone receptor-selective agonist (SRP/urocortin II)decreased food intake in fasted mice (Fig. 7) (15, 23). Becauseintermedin is specifically expressed in the muscularis mucosaelayer of the stomach, it could have a role in gastrointestinalfunctions. We therefore studied the ability of intermedin toregulate gastric emptying activity in mice. As shown in Fig. 8,intraperitoneal administration of intermedin suppressed gas-tric emptying activity, similar to treatment with a known gas-tric emptying suppression peptide, SRP/urocortin II (15, 23).Likewise, treatment with ADM also suppressed gastric empty-ing activity, but with a lower potency. Thus, intermedin couldmediate anorexic responses through the regulation of gastro-intestinal motility (22, 24).
DISCUSSION
We have used a genomic approach to study novel polypeptideligands and receptors based on the evolutionary conservation ofpolypeptides (14, 15, 25, 26). Based on the analysis of theevolution of calcitonin/CGRP family ligands from diverse ver-tebrates, we have identified a novel family peptide (intermedin)that is expressed in the pituitary and digestive tract. Studiesusing 293T cells expressing recombinant CRLR and RAMPsdemonstrated that intermedin is a bioactive peptide and acti-vates Gs of the G protein family through CRLR/RAMP receptorcomplexes. In contrast to CGRP and ADM, which exhibited apreferential stimulation of CRLR when co-expressed withRAMP1 and RAMP2 or RAMP3, respectively, intermedin rep-resents a nonselective agonist for the three CRLR/RAMP re-ceptor complexes.
Since the discovery of calcitonin in the 1960s, the calcitonin/CGRP family peptides have been studied extensively. As aresult of gene duplication and functional divergence, this groupof peptide hormones acts on diverse systems. Coupled with twoclosely related GPCRs and three unique RAMPs that transportreceptors to the cell surface, a complex receptor/ligand signal-ing system operates in diverse vertebrates (5). Calcitonin,CGRP, ADM, and amylin are expressed in a tissue-specificmanner, with the highest expression in thyroid C cells, thecentral nervous system, adrenal glands, and islet B cells, re-spectively. Although it has been recently established that thesignaling by CGRP, ADM, and amylin is unique among peptidehormones and requires the formation of a receptor/RAMP com-plex, the exact role of these peptides and their cognate recep-tors in different physiologies remains to be investigated. Thepresent discovery of intermedin as a calcitonin/CGRP familypeptide highly expressed in the pituitary and digestive tractprovides a new ligand for peripheral regulation mediated bythe CRLR/RAMP system. As a first step in defining the role ofCRLR/RAMP receptor complexes in intermedin signaling, weinvestigated the activation of CRLR/RAMP receptor complexesin transfected 293T cells and demonstrated that RAMP is re-quired for mediating intermedin action through CRLR. Of in-
FIG. 5. Expression of intermedin indigestive tissues. For analysis of inter-medin mRNAs in the human digestivesystem, normalized first strand cDNApreparations from the human esophagus,stomach, duodenum, jejunum, ileum, ileo-cecum, cecum, ascending (Asc.) colon,transverse (Trans.) colon, descending(Des.) colon, and rectum (first panel, 1 ng oftemplate/reaction; second panel, 10 pg oftemplate/reaction) were obtained fromClontech (A). Specific bands (303 bp) werePCR-amplified using intermedin gene-specific primer pairs under high stringencyconditions. The primer sequences for inter-medin PCR analysis are 5�-AGGGAGGG-GAACTCAGCAGTTCAGGAG-3� (forward)and 5�-GTTCTTGTTCTTGCTGTCACTT-GGGCCT-3� (reverse). Expression of glyc-eraldehyde-3-phosphate dehydrogenase(GAPDH) transcripts in different cDNAtemplates was also analyzed to assess thequality of the cDNA templates (third panel,1 ng of template/reaction; fourth panel, 10pg of template/reaction). Immunohisto-chemical staining of mouse stomach sec-tions showed that intermedin was foundprimarily in the muscularis mucosae layerof the stomach (B), and the signal was abol-ished by presaturation with the intermedinantigen (C). Specific signals are indicatedby arrows. MU, mucosal layer; MS, muscu-laris layer; SL, serosal layer.
Intermedin Is a Novel Calcitonin/CGRP Family Peptide 7271
terest, intermedin exhibited a receptor activation profile dis-tinct from that of CGRP or ADM, suggesting that intermedincould be important for select CRLR/RAMP-mediated physiolog-ical processes. It has been shown that the receptor activationprofiles of CGRP and ADM in native tissues are affected byendogenous RAMPs present in different systems. Future stud-ies on the interaction between intermedin and CRLR/RAMPreceptor complexes in different cell types and native tissues areneeded to clarify the importance of different RAMPs in inter-medin physiology. Furthermore, it has been demonstrated thateven though CGRP and ADM overlap in receptor interaction,each of these peptides apparently binds to unique bindingpockets (5); therefore, future studies on the structure-functionrelationship of intermedin and related peptides are essentialfor the characterization of the CRLR/RAMP-associated signal-ing system.
Among calcitonin/CGRP family peptides, ADM is mainly
FIG. 6. Decrease in systemic blood pressure and increase inheart rate by intermedin and related peptides. A, dose-dependentsuppression of systolic blood pressure by IMDL, IMDS, and ADM inmale Sprague-Dawley rats. Blood pressure change was monitored for 40min, and averages at 10, 20, and 30 min are presented. B, increase inheart rate upon treatment with different doses of IMDL, IMDS, andADM in male Sprague-Dawley rats. C, blockage of the hypotensiveeffect of intermedin by the CGRP receptor antagonist CGRP-(8–37) andthe putative intermedin receptor-binding domain peptide IMD-(17–47).D, suppression of blood pressure in male SHRs by intermedin andblockage of intermedin effects by receptor antagonists.
FIG. 7. Suppression of food intake by intermedin in fastedmice. Shown is the cumulative food intake in mice treated with phos-phate-buffered saline (n � 29), ADM (100 nmol/kg; n � 20), IMDS (100nmol/kg; n � 16), IMDL (100 nmol/kg; n � 17), or the type II cortico-tropin-releasing hormone receptor-selective agonist SRP/urocortin II(100 nmol/kg; n � 10) (15, 23) at 1, 2, and 4 h after treatment.
FIG. 8. Suppression of gastric emptying activity by interme-din. Shown is the reduction of gastric emptying by IMDL (100 nmol/kg;n � 24), IMDS (100 nmol/kg; n � 20), ADM (100 nmol/kg; n � 27), andSRP/urocortin II (100 nmol/kg; n � 10) at 90 min after hormone treat-ment compared with control animals receiving saline injections (n �31). Gastric emptying was calculated by comparing the stomach weightof treated mice with the stomach weight of control mice receiving nohormone treatment and killed at the time of hormone injection. Addi-tional animals injected with saline and killed at the same time ashormone-treated animals were used as experimental controls. *, signif-icantly different from control animals injected with saline alone(p � 0.05).
Intermedin Is a Novel Calcitonin/CGRP Family Peptide7272
characterized as a hypotensive hormone (27–29), whereasCGRP is important for sensory neurotransmission (3, 9–11, 13,30, 31). In addition, ADM inhibits bronchial constriction andacts as a neurohormone to inhibit water drinking and saltappetite (27, 32, 33). Studies using mutant mice suggest thatADM is indispensable for vascular morphogenesis during em-bryonic development (7, 8), whereas �-CGRP is important forthe modulation of sympathetic activity and inflammatory reac-tions (9–11). Therefore, CRLR in different tissues could medi-ate the actions of multiple paralogous ligands, and the physi-ological role of this receptor is partly dependent on activatingligands derived from neighboring cells and/or the general cir-culation. Because intermedin interacts with CRLR/RAMP re-ceptor complexes, the known receptors for CGRP and ADM,intermedin could regulate diverse physiological functions thathave been attributed to ADM or CGRP. As demonstrated inthis study, intermedin decreases blood pressure in both normalrats and SHRs as effectively as the better characterized ADMand CGRP, suggesting that intermedin could regulate vascu-lature homeostasis (27). Immunohistochemistry studies haveshown that CRLR and RAMPs are found in the entire vascu-lature and that CRLR is expressed mainly in the endotheliallayer (34); therefore, intermedin and related peptides decreaseblood pressure via the activation of CRLR/RAMP receptor com-plexes in vascular endothelial cells. Concomitant with a hypo-tensive effect, intermedin treatment also increases heart rate.The increase in heart rate by intermedin and related peptidescould be a reflex response to the hypotensive effect; however,the exact mechanisms remain to be investigated because theCRLR gene has been shown to be expressed in cardiac myo-cytes (35) in addition to the cardiac vasculature (34, 36). Fur-thermore, intermedin could have cardioprotective and anti-bronchial constriction activities that are important for theregulation of cardiac and respiratory homeostasis (27, 37). Be-cause intermedin is not detected in proximity to the vascula-ture system using immunohistochemical analysis, furtherstudies are needed to reveal whether intermedin represents anendocrine hormone involved in the regulation of cardiac andvasculature systems.
Earlier studies on CGRP and ADM have shown that thesepeptides and the CRLR signaling system play an importantrole in gastrointestinal functions, including motility and secre-tions from the stomach and colon (38, 39). Similar to earlierstudies on CGRP and ADM, exogenous intermedin administra-tion was found to exhibit an anorexic effect and to suppressstomach emptying responses in mice. These data suggest thatintermedin could have roles in the regulation of energy balancevia a paracrine mechanism; however, it is possible that theobserved effect on feeding behavior is secondary to alterationsin gastric motility. Because intermedin is expressed in multiplegastrointestinal tissues, intermedin could have additional rolesin the gastrointestinal system that remain to be characterized.In support of this view, it has been shown that CRLR is ex-pressed in columnar cells lining the secretory ducts of theparotid gland and in capillaries and venules of the esophagus(34).
The observation that intermedin is expressed in the anteriorand intermediate lobes of the pituitary points to a potential rolefor intermedin and the CRLR/RAMP signaling system in theregulation of pituitary hormone secretion. Although the role ofintermedin in the regulation of pituitary hormone secretionwas not examined specifically in this study, earlier studies onADM have shown that administration of ADM increases circu-lating prolactin levels in humans (40, 41). Therefore, it ispossible that intermedin may play a role in the regulation ofpituitary functions. Further studies on the exact expression
pattern of intermedin in the pituitary and other tissues duringdevelopment are important for the understanding of interme-din physiology.
Earlier studies on the evolution of peptide hormones haveshown that selection pressure has favored the conservation offunctionally important or mature regions of polypeptide hor-mone precursors. The finding that only the C-terminal end ofthe intermedin precursor was conserved during evolution sug-gested that the C-terminal sequences of the intermedin precur-sors represent the mature peptide and further strengthenedthe theory that sequence conservation among species providesimportant information on the functional characteristics of genesequences. Of interest, comparative sequence studies of inter-medin precursors from different vertebrates showed that theN-terminal cleavage sites of putative mature intermedins varyin position, whereas a downstream arginine residue is com-pletely conserved in all species studied. These data indicatethat mature intermedin from diverse species could be of vary-ing lengths and that a shorter human intermedin (e.g. IMDS)could be generated after post-translational processing at thedownstream basic residue. Future studies on human samplesare necessary to reveal the exact mature form(s) of humanintermedin. Furthermore, the recent availability of multiplesequenced vertebrate genomes will allow the identification andcharacterization of additional peptide hormones on a globalscale based on the genomic profiling approach we used to iden-tify intermedin and other novel peptide hormones (15).
In conclusion, we have identified and characterized a novelcalcitonin/CGRP family gene and demonstrated that encodedintermedin peptides are biologically active in diverse in vitro andin vivo CRLR/RAMP assays. Therefore, intermedin is a physio-logical regulator of gastrointestinal, cardiovascular, and otherbioactivities mediated by the CRLR/RAMP receptor complexes.Although the four mammalian CGRP-related peptide hormones(�-CGRP, �-CGRP, ADM, and intermedin) are capable of inter-acting with CRLR, optimal regulation by this GPCR signalingpathway likely depends on an integrated release of differentendocrine/paracrine ligands in a tissue-specific and time-coordi-nated manner. Future studies on tissue distribution and endog-enous fluctuation of intermedin under normal or pathologicalconditions are important to formulate pharmacological therapiesfor diverse pathological conditions in cardiovascular, pulmonary,gastrointestinal, and neuroendocrine systems.
Acknowledgments—We thank Augustin Sanchez and Caren Spencerfor technical and editorial assistance. We gratefully acknowledge Drs.Aaron J. W. Hsueh, Anita Payne, Linda Giudice, and Mary Lake Polan(Department of Obstetrics and Gynecology, Stanford University Schoolof Medicine) for providing support and encouragement for these andrelated efforts.
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Intermedin Is a Novel Calcitonin/CGRP Family Peptide7274
Jaesook Roh, Chia Lin Chang, Alka Bhalla, Cynthia Klein and Sheau Yu Teddy HsuReceptor Complexes
through the Calcitonin Receptor-like Receptor/Receptor Activity-modifying Protein Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family Peptide Acting
doi: 10.1074/jbc.M305332200 originally published online November 13, 20032004, 279:7264-7274.J. Biol. Chem.
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