Proc. Natl. Acad. Sci. USA
`Vol. 96, pp. 1569–1573, February 1999
`Medical Sciences
`
`Prototypic G protein-coupled receptor for the intestinotrophic
`factor glucagon-like peptide 2
`DONALD G. MUNROE*†, ASHWANI K. GUPTA*, FATEMEH KOOSHESH*, TEJAL B. VYAS*, GEIHAN RIZKALLA*,
`HONG WANG*, LIDIA DEMCHYSHYN*, ZHI-JIE YANG*, RAJENDER K. KAMBOJ*, HONGYUN CHEN*,
`KIRK MCCALLUM*, MARTIN SUMNER-SMITH*‡, DANIEL J. DRUCKER§, AND ANNA CRIVICI*¶
`*Allelix Biopharmaceuticals Inc., 6850 Goreway Drive, Mississauga, Ontario L4V 1V7, Canada; and §Department of Medicine, The Toronto Hospital, Banting and
`Best Diabetes Centre, University of Toronto, Toronto, Ontario M5G 2C4, Canada
`
`Edited by Donald F. Steiner, The University of Chicago, Chicago, IL, and approved November 18, 1998 (received for review August 18, 1998)
`
`Glucagon-like peptide 2 (GLP-2) is a 33-aa
`ABSTRACT
`proglucagon-derived peptide produced by intestinal enteroen-
`docrine cells. GLP-2 stimulates intestinal growth and up-
`regulates villus height in the small intestine, concomitant with
`increased crypt cell proliferation and decreased enterocyte
`apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia
`resulting from total parenteral nutrition. However, the mech-
`anism underlying these actions has remained unclear. Here we
`report the cloning and characterization of cDNAs encoding rat
`and human GLP-2 receptors (GLP-2R), a G protein-coupled
`receptor superfamily member expressed in the gut and closely
`related to the glucagon and GLP-1 receptors. The human
`GLP-2R gene maps to chromosome 17p13.3. Cells expressing
`the GLP-2R responded to GLP-2, but not GLP-1 or related
`peptides, with increased cAMP production (EC50 5 0.58 nM)
`and displayed saturable high-affinity radioligand binding (Kd
`5 0.57 nM), which could be displaced by synthetic rat GLP-2
`(Ki 5 0.06 nM). GLP-2 analogs that activated GLP-2R signal
`transduction in vitro displayed intestinotrophic activity in vivo.
`These results strongly suggest that GLP-2, like glucagon and
`GLP-1, exerts its actions through a distinct and specific novel
`receptor expressed in its principal target tissue, the gastro-
`intestinal tract.
`
`Glucagon-like peptides (GLPs) encoded by the proglucagon
`gene play key roles in glucose homeostasis, gastric emptying,
`insulin secretion, and appetite regulation (1). Glucagon and
`GLP-1 exert their effects through distinct G protein-coupled
`receptors (GPCRs). In contrast, unique receptors for GLP-2,
`glicentin, and oxyntomodulin have not yet been identified,
`despite considerable attempts at receptor isolation via classical
`molecular biology approaches (2). Recent studies have shown
`that GLP-2 is a potent intestinal growth factor that stimulates
`crypt cell proliferation and inhibits epithelial apoptosis (3).
`GLP-2 promotes epithelial proliferation in both small and
`large intestine; however, the mechanisms utilized by GLP-2 for
`promotion of intestinal growth remain unclear.
`To understand the mechanisms underlying GLP-2 action, we
`have carried out studies directed at the identification and
`cloning of the putative GLP-2 receptor. We now have isolated
`rat and human cDNAs encoding GLP-2-responsive GPCRs,
`which show highest similarity to receptors for glucagon and
`GLP-1. The GLP-2R is coupled to activation of adenylate
`cyclase, and the receptor is expressed selectively in rat hypo-
`thalamus and the gastrointestinal tract, known targets of
`GLP-2 action. These findings establish GLP-2 as a novel
`hormone that, like glucagon and GLP-1, exerts its actions
`through a distinct receptor expressed in a highly tissue-
`restricted manner. The GLP-2R should provide an important
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked ‘‘advertisement’’ in
`accordance with 18 U.S.C. §1734 solely to indicate this fact.
`PNAS is available online at www.pnas.org.
`
`target for isolation of small molecules mimicking GLP-2 action
`and for future studies delineating specific mechanisms under-
`lying GLP-2 action in the gut and central nervous system.
`
`Methods and Materials
`Primers, cDNA Libraries, and Cloning Strategy. Initial
`attempts at low-stringency hybridization of intestine and brain
`cDNA libraries using GLP-1RyGlucagonR cDNA sequences
`were not successful. Two million cDNA clones from rat
`hypothalamus and rat duodenumyjejunum cDNA libraries
`subsequently were screened with degenerate oligonucleotides
`derived from conserved transmembrane II and VII GPCR
`coding sequences: C4–4 (59-AACTACATCCACMKGM
`AYCTGTTYVYGTCBTTCATSCT-39) (IUB nomenclature)
`and C9 –2R (59-TCYRNCTGSACCTCMYYRTTGAS-
`RAARCAGTA-39) (for nomenclature, see ref. 4). First-round
`cDNA plugs (1,057) were isolated in this screen. In a comple-
`mentary strategy, PCR was conducted on intestinal cDNA
`templates by using sets of degenerate PCR primers, based on
`conserved transmembrane amino acid motifs from family B
`GPCRs or from motifs conserved mainly within the glucagony
`glucose-dependent insulinotropic polypeptide (GIP)yGLP-1
`receptor subfamily. PCR products were Southern-blotted and
`probed with 32P-end-labeled C4–4 oligonucleotide. PCRs,
`amplified from rat neonatal
`intestine cDNA (Stratagene;
`catalog no. 936508) were chosen for cloning. These products
`had been amplified with the degenerate primers M2F (59-
`TTTTTCTAGAASRTSATSTACACNGT SGGCTAC-39 )
`(based on conserved transmembrane domain I sequences) and
`M7R (59-TTTTCTCGAGCCARCARCCASSWRTART-
`TGGC-39) (based on conserved transmembrane III sequenc-
`es). PCR products were cloned into pBluescript, screened by
`filter hybridization with the nested C4-4 oligonucleotide, and
`sequenced, leading to the identification of a sequence frag-
`ment from a novel GPCR family B member, designated WBR,
`that ultimately proved to be the GLP-2R. Two new GLP-2R-
`specific PCR primers, P23-F1 (59-TCTGACAGATATGA-
`CATCCATCCAC-39) and P23-R1 (59-TCATCTCCCTCT-
`TCTTGGCTCTTAC-39), were used to screen the 1,057 cDNA
`plugs obtained by hybridization screening,
`leading to the
`
`This paper was submitted directly (Track II) to the Proceedings office.
`Abbreviations: EBNA, Epstein–Barr nuclear antigen; GPCR, G pro-
`tein-coupled receptor; GLP-1 and -2, glucagon-like peptide 1 and 2,
`respectively; GLP-2R, GLP-2 receptor; GIP, glucose-dependent insu-
`linotropic polypeptide.
`Data deposition: The sequences reported in this paper have been
`deposited in the GenBank database (accession nos. AF105367 and
`AF105368).
`†To whom reprint requests should be addressed. e-mail: dmunroe@
`allelix.com.
`‡Present address: Base4 Bioinformatics, 6299 Airport Road, Missis-
`sauga, Ontario L4V 1N3, Canada.
`¶Present address: Ligard Pharmaceuticals, Inc. 10275 Science Center
`Drive, San Diego, CA 92121.
`
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`FIG. 1. Multiple alignment of the human (GL2RoHUMAN) and rat (GL2RoRAT) GLP-2R amino acid sequences with human GLP-1, GIP,
`and glucagon receptor sequences. Known receptor sequences are designated by Swiss-Prot identifiers. Alignment was performed with CLUSTAL W
`1.60 and rendered with GENEDOC 2.2. Identities of rat and human GLP-2R sequences is shown in gray, and identities across all five receptor members
`are indicated by black shading. A predicted signal-peptide cleavage site in human and rat GLP-2R is indicated by an inverted triangle. Six conserved
`cysteine residues are indicated by arrows. Seven predicted transmembrane domains are shown as solid, black boxes labeled with Roman numerals,
`and asterisks are shown for spacing every 20 aa. The GenBank accession numbers for the human and rat GLP-2R sequences are AF105367 and
`AF105368, respectively.
`
`identification of three independent clones, two from the
`duodenumyjejunum library and a third from hypothalamus,
`which, together, contained a 2,537-bp cDNA insert encoding
`full-length rat GLP-2R.
`To clone the human GLP-2R, the coding region of the rat
`GLP-2R was used to screen a human hypothalamus cDNA
`library (CLONTECH; catalog no. 1172a), from which a single
`positive clone (HHT13) was isolated and sequenced. A full-
`length insert was ligated into pcDNA3 for expression studies.
`Intestinotrophic Activity, cAMP Determination, and Radi-
`oligand-Binding Studies. For cAMP assays, an episomal stable
`
`cell line was prepared by lipofection of 293-EBNA (Epstein–
`Barr nuclear antigen) cells (Invitrogen) with a pREP7-based
`(Invitrogen) construct containing the Met-42 3 Ile-550 ORF
`of rGLP-2R. Parental 293-EBNA cells, as well as the stable cell
`line rG2R, expressed receptors for vasoactive intestinal
`polypeptide and pituitary adenylate cyclase-activating
`polypeptide. Therefore, the ligand specificity of GLP-2R was
`tested further in transiently transfected COS cells, which
`expressed no functional receptors for any of the ligands tested.
`For cAMP assays, cells were treated at 80% confluency with
`GLP-2 peptide analogs at concentrations ranging from 10212
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`to 1025 M for 30 min in medium containing 3-isobutylmeth-
`ylxanthine. The reaction was terminated with the addition of
`95% ethanol and 5 mM EDTA. Aliquots of the ethanol extract
`were used to determine cAMP levels using an enzyme immu-
`noassay kit (Amersham) as described by the manufacturer.
`Results were analyzed with GRAPHPAD PRISM software and
`expressed as pmol cAMP per well. For radioligand-binding
`assays, cells expressing GLP-2R were harvested and homog-
`enized in 25 mM Hepes (pH 7.4) buffer containing 140 mM
`NaCl, 0.9 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2, 17 mg/ml
`Diprotin A, and 100 mM phenanthroline. Homogenates were
`centrifuged for 10 min at 1,000 3 g at 4°C to remove cellular
`debris. For saturation experiments, membranes containing 25
`mg protein were incubated with increasing concentrations of
`125I-[Tyr-34]GLP-2 (5–2,000 pM final concentration) in a
`volume of 0.5 ml for 2 hr at 4°C. Nonspecific binding was
`determined by the addition of 10 mM of native rat GLP-2 and
`subtracted from total binding to estimate specific binding to
`GLP-2R. Parallel experiments confirmed the lack of specific
`binding when the GLP-2R expression construct was not used.
`For competition-binding experiments, assays were initiated by
`the addition of 200 pM (final concentration) of 125I-
`[Tyr34]GLP-2 with increasing concentrations of competing
`peptide analogs (10211 to 1025 M) for 2 hr as described above.
`Reactions were terminated by centrifugation at 13,000 3 g for
`15 min at 4°C. The pellets were washed three times with cold
`50 mM Tris buffer, and radioactivity was quantitated in a
`gamma counter. Results were analyzed by GRAPHPAD PRISM
`software.
`Intestinotrophic activities of various peptide analogs were
`determined by assessment of small bowel weight as described
`(5), after 14-day treatments with 2.5 mg of test peptide or PBS
`(vehicle-treated control) administered twice daily. Activity
`was defined as follows: active, small bowel wet weight 40–70%
`greater than in vehicle-treated control animals; partially active,
`20–40% greater than controls; inactive, less than 20% greater
`than controls.
`RNAse Protection Assay. A fragment of GLP-2R cDNA was
`subcloned into pBluescript (Stratagene) for in vitro transcrip-
`tion with T3 or T7 RNA polymerases. The probe, called F1,
`spanned nucleotides encoding amino acids Met-1 3 Arg-210.
`RNase protection assay was carried out essentially as described
`(6), using 50 mg of total RNA from adult rat tissues or 50 mg
`of yeast tRNA (negative control) or tRNA spiked with a
`known copy number of sense-strand cRNA (for standard curve
`construction). Each sample was hybridized with 100,000 cpm
`of [32P]CTP-labeled antisense cRNA and then digested with
`RNases T1 (140 unitsyml) and A (8 mgyml) at 30 C for 1 hr.
`The deproteinized, ethanol-precipitated probe was run on a
`5% sequencing gel and analyzed after PhosphorImaging (Mo-
`lecular Dynamics) with IMAGEQUANT software. RNA copy
`number was calculated by interpolation relative to the standard
`curve after taking the lengths and specific activities of undi-
`gested and digested probes into account. A second RNase
`probe from a different region of the GLP-2R cDNA was used
`to confirm the quantitative results (data not shown).
`
`RESULTS AND DISCUSSION
`GLP-2 and the peptide hormones GLP-1, glucagon, and GIP
`have closely related amino acid sequences (7). Similarly, the
`sequences of cloned receptors for the latter three peptides
`form a cluster within the parathyroid hormone receptor-like
`GPCRs family B (8–11), suggesting that the GLP-2 receptor
`might also be found within this subfamily. Initial attempts at
`GLP-2 receptor cloning by conventional screening of cDNA
`libraries at low stringency with a combination of GLP-1 and
`glucagon receptor cDNA probes were not successful. Accord-
`ingly, we next used a combined approach of reverse transcrip-
`tion–PCR and hybridization screening followed by expression
`
`FIG. 2. Ligand-selective and concentration-dependent cAMP re-
`sponse to rat GLP-2 in transiently lipofected COS cells. (A) Ligand
`specificity of cAMP response to GLP-2. cAMP response to peptide
`analogs of family 2 GPCR ligands was determined in COS cells
`transiently lipofected with the GLP-2R expression vector p587-70 or
`the parental pcDNA3 expression vector. GLP-2 concentration was 1
`nM; all other peptides were used at 10 nM. A similar profile of peptide
`specificity was observed with the human GLP-2R (data not shown).
`(B) Concentration–response curve for cAMP accumulation in re-
`sponse to synthetic rat GLP-2 in rG2R cells stably expressing GLP-2R.
`
`analysis of candidate cDNAs. As described in Methods and
`Materials, this strategy resulted in the isolation of a 2,537-bp rat
`GLP-2R cDNA insert encoding a 550-aa putative family B
`GPCR (Fig. 1). Hydropathy analysis of the GLP-2R amino acid
`sequence revealed a typical 7-transmembrane topology plus a
`hydrophobic amino-terminal signal peptide (data not shown).
`The GLP-2R gene product belongs to the GLP-1yglucagony
`GIP receptor gene subfamily. Conserved features include a
`possible signal-peptide cleavage site between Val-64 and Thr-
`65, potential N-glycosylation sites within the amino-terminal
`putative extracellular domain, and six cysteine residues con-
`served in the mature GLP-1, GIP, and glucagon receptor
`amino-terminal domains (Fig. 1). Two putative alternative
`translation initiation codons, Met-1 and Met-42, were found
`amino-terminal to the first transmembrane domain in the rat
`GLP-2R. Functional analysis of Met-1 and Met-42 site-
`directed mutants showed they were functionally identical
`(unpublished results), consistent with signal-peptide removal
`predicted to yield an identical 486-aa mature polypeptide.
`Overlapping regions of the hypothalamus and duodenumy
`jejunum cDNA clones encoded homologous polypeptide frag-
`ments. Moreover, no evidence was obtained for differential
`splicing of intestinal GLP-2R RNA from an RNase protection
`assay employing two nonoverlapping probes derived from
`GLP-2R cDNA, which together spanned 387 of the 550
`GLP-2R codons (data not shown). Additionally, sequencing of
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`
`GLP-2-binding sites on these cells was shown by using a radio-
`iodinated, C-terminally extended GLP-2 analog, 125I-[Tyr-
`34]GLP-2 (Fig. 3A). From Scatchard analysis of the saturation
`isotherms, a Bmax value of 1,839 fmolymg of protein and a Kd of
`0.57 nM were obtained for the radioligand. Mock-transfected
`cells showed no specific binding to the radioligand (data not
`shown). Competition-binding studies with rat GLP-2 revealed a
`high-affinity site (Ki 5 0.06 nM) and a low-affinity site (Ki 5 259
`nM) (Fig. 3B). In contrast, no high-affinity GLP-1 sites were
`observed in the transfected GLP-2RyrG2R clone. Ki values
`determined for GLP-1, glucagon, and GIP peptides were 928,
`500, and 765 nM, respectively. Although the binding and Scat-
`chard data may reflect, in part, a degree of receptor overexpres-
`sion, the functional studies and binding data provide firm evi-
`dence for a cDNA that encodes a functional high-affinity, ligand-
`selective GLP-2 receptor.
`The human GLP-2R polypeptide showed 81.6% similarity to
`rat GLP-2R (Fig. 1). Functional expression of the cloned human
`GLP-2R conferred a functional response to GLP-2 and the
`appearance of high-affinity ligand-binding sites on 293-EBNA
`cells, similar to data obtained with the rat GLP-2R (data not
`shown). Furthermore, the cloned human GLP-2 receptor exhib-
`ited the same profile of peptide-binding specificity (Fig. 2A and
`unpublished data) as the rat receptor. The gene encoding human
`GLP-2R was identified by screening an arrayed BAC library of
`human genomic DNA (Genome Systems, St. Louis), confirmed
`by sequencing, and mapped to chromosome 17p13.3 by fluores-
`cence in situ hybridization analysis (data not shown).
`A quantitative ribonuclease protection assay method was
`used to determine the tissue distribution of rat GLP-2R RNA
`because no signals were detected on multitissue Northern
`blots. GLP-2R RNA levels were highest in jejunum, followed
`by duodenum, ileum, colon, and stomach, whereas expression
`was undetectable in seven other tissues (Table 1). This expres-
`sion pattern is clearly concordant with previously reported
`functional responses to GLP-2 in duodenum (12, 13), jejunum,
`ileum (5, 12, 14, 15), and colon (12, 16, 17); in contrast, no
`proliferative or histological changes were seen after GLP-2
`treatment in spleen, heart, kidney, lung, or brain (18). Thus,
`GLP-2R expression is detected in known GLP-2 target tissues.
`This observation, together with the functional data from
`
`Table 1. Quantitative GLP-2R RNA distribution in various rat
`tissues determined by RNase protection
`b-Actin
`F1 quantitation,
`quantitation,
`GLP-2Ry-actin,
`copies per mg
`copies per mg
`ratio
`total RNA
`total RNA
`Tissue
`76.8 3 1025
`15,500,000
`11,900
`Jejunum
`10.7 3 1025
`85,700,000
`9,150
`Duodenum
`14.6 3 1025
`51,400,000
`7,490
`Ileum
`21.0 3 1025
`19,800,000
`4,150
`Colon
`6.48 3 1025
`23,600,000
`1,530
`Stomach
`,1.48 3 1025
`,600
`40,600,000
`Brain
`,9.09 3 1025
`,600
`6,600,000
`Heart
`,4.03 3 1025
`,600
`14,900,000
`Kidney
`,3.59 3 1025
`,600
`16,700,000
`Liver
`,1.56 3 1025
`,600
`38,500,000
`Lung
`,13.0 3 1025
`,600
`4,600,000
`Muscle
`,1.34 3 1025
`,600
`44,800,000
`Spleen
`Total RNA (50 mg) from rat tissues or sense-strand cRNA standards
`was hybridized to radiolabeled antisense cRNA probes prepared in
`vitro from GLP-2R cDNA (F1) or actin cDNA. After RNase digestion
`as described in Methods and Materials, protected probe was precipi-
`tated, electrophoresed, and quantitated by PhosphorImage analysis
`relative to the standard curve obtained from sense-strand cRNA. RNA
`quantitation is expressed as copy number per mg of total RNA.
`GLP-2R RNA copy number was detectable to a lower limit of 30,000
`copies per 50 mg sample, setting the limit of detection shown above for
`nongastrointestinal tissues.
`
`FIG. 3. Binding of 125I -[Tyr-34]GLP-2 to cell membranes prepared
`from rG2R cells stably transfected with GLP-2R cDNA. (A) Satura-
`tion isotherms of the specific binding of 125I -[Tyr-34]GLP-2 to
`membranes. Results shown are representative of six independent
`experiments, each conducted in triplicate. From Scatchard analysis
`(Inset), maximal-binding Bmax was estimated at 1,839 fmolymg protein,
`and a Kd value of 0.57 nM was obtained. (B) Competition binding of
`125I-[Tyr-34]GLP-2 binding to cell membranes in the presence of
`unlabeled peptides. Data are shown for the concentration-dependent
`inhibition of 125I-[Tyr-34]GLP-2 binding (200 pmol) to GLP-2R by
`various peptide analogs from at least two independent experiments
`conducted in triplicate. Inhibitory constants (Ki) were estimated by
`using GRAPHPAD PRISM.
`
`the full-length human GLP-2R cDNAs confirmed the identity
`of hypothalamic and gastrointestinal GLP-2Rs (Fig. 1).
`To assess whether the predicted GLP-2R sequence encodes
`a functional GLP-2 receptor, a GLP-2R expression construct,
`p587-70, was transiently transfected into COS cells. Because
`related family B GPCRs show functional coupling to cAMP
`production mediated by the heterotrimeric G protein Gs,
`cAMP accumulation was measured after incubation with
`GLP-2. Treatment of GLP-2R-transfected COS cells with 1
`nM GLP-2 resulted in a 4-fold rise in cAMP levels relative to
`untreated cells, approximately equal to the response seen with
`10 mM forskolin (Fig. 2A). Treatment with 10 nM GLP-1,
`glucagon, GIP, exendin-3, and seven other family B GPCR
`ligands did not
`induce cAMP production in p587-70-
`transfected cells. Control cells transfected with vector DNA
`alone failed to respond to any of the peptides tested, including
`GLP-2, though forskolin did induce cAMP production.
`With a stable GLP-2R episomal expression cell line, rG2R,
`greater than 20-fold cAMP induction routinely was achieved with
`1 or 10 nM GLP-2 (data not shown), confirming the potent
`induction of cAMP accumulation by GLP-2. In contrast, the
`nontransfected 293-EBNA cells showed no response to GLP-2.
`The EC50 of the cAMP response to GLP-2 in rG2R cells was 0.58
`nM (Fig. 2B). The presence of saturable, specific, ligand-selective
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`Table 2.
`
`In vitro and in vivo activity profiles of selected peptide analogs of GLP-2
`Ki, nMa*
`
`In vivo
`activity§
`Active
`Partially active
`Inactive
`Active
`Active
`Inactive
`Inactive
`Partially active
`Inactive
`Active
`Active
`Inactive
`Inactive
`Inactive
`
`Low-afinity
`High-affinity
`Peptide
`259 6 46
`0.06 6 0.00
`rGLP-2(1-33)¶
`140 6 2
`—
`N-Ac-rGLP-2(1-33)
`[Arg-1]rGLP-2(21-33)
`n*
`NA
`n§
`ND
`[Arg-34]rGLP-2(1-34)
`255 6 7
`0.56 6 0.3
`[Tyr-34]hGLP-2
`876 6 147
`—
`rGLP-2(2-33)
`251 6 8
`—
`rGLP-2(3-33)
`584 6 19
`0.30 6 0.00
`rGLP-2(1-29)
`977 6 470
`—
`[Thr-7 insertion]
`126 6 5
`—
`[Gly-2]GLP-2(1-33)
`596 6 9
`1.7 6 0.4
`hGLP-2(1-33)¶
`500 6 332
`—
`Glucagon
`928 6 1
`—
`GLP-1(7-36)amide
`765 (n 5 1)
`—
`GIP
`NA, not active—no detectable binding; ND, not determined.
`*n 5 2, except where indicated.
`†n 5 3.
`‡Relative to 100 nM rGLP-2(1-33), n 5 3.
`§Relative to vehicle-treated control animals, n 5 4 or greater. In vivo activity is based on changes in small bowel wet weight after 14-day treatment
`as described in Methods and Materials.
`¶rGLP-2(1-33) is native rat GLP-2 peptide; hGLP-2(1-33) is native human GLP-2 peptide.
`
`EC50, nM†
`1.00 6 0.2
`20.8 6 0.1
`901 6 41
`3.1 6 0.3
`1.4 6 0.1
`210 6 22
`10.7 6 0.8
`3.60 6 0.4
`1,100 6 30
`2.0 6 0.2
`1.3 6 0.20
`NA
`NA
`NA
`
`Emac, %†‡
`100 6 0
`80.1 6 7
`69.0 6 2
`105 6 10
`113 6 5
`109 6 8
`115 6 5
`102 6 8
`76 6 4.0
`103 6 6
`99 6 10.6
`NA
`NA
`NA
`
`experiments with cloned GLP-2R cDNA, suggests that this
`receptor mediates the intestinotrophic actions of GLP-2.
`Pharmacological support for this hypothesis was obtained from
`parallel in vivoyin vitro studies of GLP-2 analogs containing
`simple changes in sequence and length (Table 2). Carboxyl-
`terminal extension analogs bound and activated GLP-2R and
`retained in vivo activity whereas those with amino-terminal
`extensions lost both activities. Analogs with blocked amino- or
`carboxyl-terminal residues displayed diminished in vivo activity
`and GLP-2R activation. Insertion of a Thr residue between
`GLP-2 residues 6 and 7 resulted in loss of activity in vivo and in
`vitro. Truncation of the carboxyl-terminus to a 29-residue peptide
`(analogous to glucagon) reduced but did not eliminate in vivo or
`in vitro activities. Interestingly, truncation of one or two amino-
`terminal residues abolished in vivo activity but did not completely
`eliminate binding or the GLP-2R cAMP response. Taken to-
`gether, a clear correspondence was revealed between the struc-
`tural requirements for GLP-2R binding and activation and the in
`vivo intestinotrophic activity of GLP-2, providing additional
`evidence that the GLP-2R isolated here and the intestinotrophic
`GLP-2 receptor mediating GLP-2 action in vivo are synonymous.
`Enteroglucagon synthesis long has been associated with a
`humoral adaptive response to massive small bowel resection, in
`which hyperplasia and elongation of jejunal villi are seen (19–22).
`Proglucagon-derived GLP-2 is detectable in plasma from fasted
`rats and humans and rises 1.5- to 3.6-fold after feeding (23).
`Moreover, the intestinotrophic efficacy of GLP-2 has been shown
`after administration by i.p., i.m., or s.c. routes (14), as well as by
`coinfusion in parenterally fed rats (12). Thus, it is likely that
`circulating GLP-2 mediates adaptive changes in the villus-
`absorptive area in the small intestine. The cloning and charac-
`terization of a GLP-2 receptor expressed in the gastrointestinal
`tract qualifies GLP-2, like GLP-1, glucagon, and GIP, as a bona
`fide endocrine hormone and should facilitate the discovery of
`novel pharmacologic agents with similar functional activity. The
`expression of GLP-2R in hypothalamus also raises the possibility
`of as yet undescribed role(s) for this intestinotrophic hormone in
`the central nervous system.
`
`We thank P. Khanna, Y.-D. Huang, R. Mathieson, Y.-P. Zhang, and
`E. Fan for technical assistance; and J. W. Dietrich, R. Zastawny, and
`D. Lee for advice and critical reading of this manuscript. D.J.D. is a
`consultant to Allelix Biopharmaceuticals Inc.
`
`10.
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