I. Introduction

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Guanine nucleotide-binding protein (G protein)-coupled receptors have been subdivided into distinct subgroups, based upon shared structural identity and evolutionary origin (Josefsson, 1999). Receptors within the family B (or family 2) subgroup, exemplified by the secretin receptor, the original family B receptor member (Ishihara et al., 1991), exhibit less homology with other GPCR¹ subfamilies and consist of three distinct subgroups, with subfamily B1 containing multiple receptors for peptide hormones (Harmar, 2001). The genes encoding the structurally related peptides (Table 1 and Fig. 1) secretin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, growth hormone releasing hormone, and glucose-dependent insulinotropic polypeptide are expressed in the gastrointestinal tract and/or brain, and signal through G_s leading to activation of adenylate cyclase and increased levels of cyclic AMP. A single proglucagon gene in mammals (Irwin, 2001) encodes three distinct structurally related peptides, glucagon, GLP-1, and GLP-2, which exhibit unique biological actions mediated by separate receptors (Bataille, 1996a,b; Drucker, 2001c). In contrast, separate receptors for glicentin, oxyntomodulin, and miniglucagon, biologically active peptides derived from the identical proglucagon precursor, have not yet been identified (Drucker, 2001c, 2002). Several members of the secretin peptide family, including secretin, GLP-1(7-36amide) and growth hormone-releasing hormone (GHRH) are amidated at the carboxyl terminus, however amidation is not always an invariant requirement for biological activity, as the nonamidated GLP-1(7-37) is equipotent with GLP-1(7-36amide) (Orskov et al., 1993). Similarly, GHRH, GLP-1, glucose-dependent insulinotropic peptide (GIP), and GLP-2 are excellent substrates for the enzyme dipeptidyl peptidase IV, which inactivates these peptides following cleavage at the position 2 alanine or proline (De Meester et al., 1999).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Family tree of selected members of family B G protein-coupled receptors, as described by Harmar, 2001, and kindly provided by Dr. A. Harmar.

Consistent with the structure of multiple G protein-coupled receptors within class 2, the secretin receptor family contains a disulfide bond linking the first and second extracellular loop domains (Asmann et al., 2000), a signal peptide, and a comparatively large extracellular domain important for ligand binding. The glucagon receptor has a region (FQG-hydr-hydr-VAx-hydr-YCFxEVQ)"hydr" being a hydrophobic and "x" any amino acidthat is highly conserved in all the members of the glucagon/secretin receptor subfamily. A highly conserved aspartic acid residue in the extracellular domain of several family B receptors has been shown to be critical for ligand binding, as exemplified by the little mouse mutation that encodes for a mutant GHRH receptor that fails to bind ligand due to replacement of the aspartic acid residue at position 7 in the extracellular domain with a glycine (Lin et al., 1993; Carruthers et al., 1994; Gaylinn et al., 1999). The nomenclature of the class 2 secretin family of receptors is comparatively straightforward, as each receptor is named for its principal and only physiologically relevant ligand, with no significant biologically meaningful cross-reactivity occurring across the spectrum of related peptide ligands and receptors (Tables 1 and 2).

	II. Secretin Receptor

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The secretin receptor is prototypic of the class II family of GPCRs, being the first member of this group of receptors to be cloned in 1991 (Ishihara et al., 1991). The concept of a circulating chemical messenger and even the introduction of the term "hormone" is related to the observation by Bayliss and Starling (1902) that a duodenal extract could stimulate pancreatic fluid secretion. That factor was subsequently purified to homogeneity and identified as a 27-residue linear polypeptide (Fig. 2) (Mutt et al., 1966) Extensive primary structure-activity studies of secretin have established that it has a diffuse pharmacophoric domain, spread throughout the length of the peptide (Ulrich et al., 1998) consistent with many of the currently recognized natural agonist ligands of the class II GPCRs.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the secretin peptide gene structure. This gene contains four exons, with the first containing 5'-untranslated sequence, the signal peptide, and a portion of the amino-terminal peptide. The second exon includes the entire mature peptide coding region with a few residues of both amino-terminal and carboxyl-terminal peptides. The third and fourth exons include portions of the carboxyl-terminal peptide, with the latter also containing 3'-untranslated sequence. COOH, the secretin carboxyl-terminal peptide; NH2, the amino-terminal peptide as described by Kopin et al. (1990, 1991).

Secretin is produced and secreted by secretory granule-containing endocrine S-cells that are scattered as single cells within the mucosal layer of the duodenum and proximal jejunum (Mutt, 1980). Major factors stimulating secretion of this hormone are luminal acid and fatty acids (Mutt, 1980), consistent with secretin actions on epithelial cells lining the pancreatic and biliary ducts, leading to the secretion of alkaline bicarbonate-rich fluid. This, in turn, helps to neutralize the acidic chyme emptied from the stomach, protecting the duodenal mucosa and providing an optimal pH for the action of bile acids and pancreatic zymogens. Secretin also slows gastric emptying to further protect the duodenum from being overwhelmed by excessively rapid delivery of acidic chyme.

The secretin receptor cDNA was first identified and cloned in 1991 (Ishihara et al., 1991). Consistent with pharmacological studies performed in the precloning era, the recombinant receptor bound secretin with high affinity and bound vasoactive intestinal polypeptide (VIP) with low affinity (Gardner et al., 1976; Ulrich et al., 1993). Potencies for stimulation of biological responses of these peptides paralleled their binding affinities. Although secretin can also bind to and activate other class II family G protein-coupled receptors (such as the VIP receptor) at low affinity, to date there have been no other subtypes of the secretin receptor identified.

Similarly, highly selective agonists that are more stable or that exhibit enhanced potency, or nonpeptidic ligands have not yet been described for the secretin receptor. The most useful antagonist of secretin action is a peptide analog of secretin having a reduced peptide bond between residues four and five [(4,5)secretin] (Haffar et al., 1991). This is consistent with primary structure-activity studies that have suggested that the selectivity of binding is most dependent on the NH₂-terminal portion of the diffuse pharmacophore, whereas the carboxyl-terminal portion further contributes to binding affinity and to biological action (Holtmann et al., 1995; Vilardaga et al., 1995).

The endogenous agonist for the secretin receptor is the linear 27-residue polypeptide, secretin (Fig. 1), secreted by endocrine S-cells in the upper small intestinal mucosa. This gastrointestinal hormone has now been isolated and sequenced in multiple animal species (Leiter et al., 1994). Minimal sequence differences are present in pig, cow, dog, rat, sheep, and human secretin, with a fully conserved NH₂-terminal domain, and only residues in positions 14, 15, and 16 exhibiting species-specific changes. Secretin in mouse and rabbit differs at residues five and six. It is only when moving to a species as divergent as the chicken that more substantial sequence differences are present, although the NH₂ terminus of chicken secretin continues to be somewhat conserved. Of note, no single species has yet been described as having more than one normally occurring form of this hormone, and no molecular variants or mutant forms of secretin have yet been described.

Consistent with the species variation of the sequence of secretin, primary structure-activity studies using hormone fragments and peptide analogs have shown that the NH₂-terminal region of the natural hormone provides key determinants for receptor selectivity, whereas its carboxyl-terminal region provides determinants for high affinity binding and biological activity (Holtmann et al., 1995; Vilardaga et al., 1995). The carboxyl-terminal region of related peptides can be fused to the secretin NH₂ terminus resulting in a chimeric peptide that still retains high affinity binding and biological activity (Park et al., 2000).

The human secretin receptor is predicted to be 440 amino acids in length, having a 21-residue signal peptide that is cleaved during biosynthesis and a deduced mature protein of 419 residues (Chow, 1995). Secretin receptors from rat and rabbit have also been cloned (Ishihara et al., 1991; Svoboda et al., 1998). The rat receptor (P23811) is 449 amino acid residues, with a 22-residue signal peptide and a 27-residue mature protein. The rabbit receptor (AF025411) is 445 amino acid residues, with a 20-residue signal peptide and a 225-residue mature protein. These sequences are 78 to 83% identical, with the major variations residing in the signal peptides and in the carboxyl-terminal tail regions.

The secretin receptor is a seven transmembrane glycoprotein having five N-linked carbohydrate groups, with four of these in the NH₂ terminus and one in the second extracellular loop region. The typical heptahelical topology of this receptor has been established using epitope tags and photoaffinity labeling by cell impermeant hydrophilic peptide probes (Dong et al., 1999a,b, 2000; Holtmann et al., 1996a, 1995). This receptor has numerous sites of potential phosphorylation on serine and threonine residues within the NH₂-terminal tail and intracellular loop regions. Secretin receptor phosphorylation has been directly demonstrated (Ozcelebi et al., 1995; Holtmann et al., 1996b), although the precise sites of modification have not been defined. There are no sites of fatty acid acylation or predicted regions having kinase activity within this receptor. A variant secretin receptor in which the third exon was spliced out to eliminate residues 44-79 from the NH₂-terminal tail, has been identified in a gastrinoma, pancreatic cancer, and pancreatic cell lines. The variant receptor functions as a dominant-negative molecule and suppresses normal secretin receptor activity, likely through formation of a heterodimer with the wild-type receptor (Ding et al., 2002a).

The secretin receptor gene is localized to human chromosome two (2q14.1) (Mark and Chow, 1995; Ho et al., 1999), spans more than 69 kb, and contains thirteen exons and twelve introns (Ho et al., 1999). The junctions between the exons interrupt at residues 24, 65, 101, 135, 168, 212, 264, 284, 307, 338, 380, and 394 (numbering of residues includes the signal peptide) (Fig. 3). The first four exons encode the critical NH₂ terminus of this receptor. Subsequent junctions are distributed throughout the loop and transmembrane regions. No alternatively spliced forms of the secretin receptor have been found to occur naturally. However, a misspliced form of this receptor that is missing exon three (leading to an in-frame deletion of residues 44 through 79 in the NH₂-terminal tail of the mature protein) was isolated from a gastrinoma in a patient with a false-negative provocative secretin stimulation test (Ding et al., 2002b). This variant form of the secretin receptor is unable to bind secretin or to signal in response to this hormone. Of particular interest, this mutant secretin receptor was shown to possess dominant-negative activity and inhibited the ability of secretin to bind and signal at a coexpressed wild-type secretin receptor (Ding et al., 2002b).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   A, structure of the mammalian proglucagon gene and the proglucagon-derived peptides liberated in distinct tissues. GRPP, glicentin-related pancreatic polypeptide; MPGF, major proglucagon-derived fragment; IP, intervening peptide. B, structural organization of the GIP precursor, as described by Tseng et al. (1993). The upper numbers represent amino acids in the rat pro-GIP sequence; lower numbers correspond to the human proGIP sequence. C, biosynthesis of GHRH. The GHRH gene includes 5 exons, and alternative untranslated exons 1 are used in different tissues (1P, placenta; 1B, hypothalamus). The GHRH mRNA encodes a prepro-GHRH precursor protein that is 103-108 amino acids, depending on the species. This is processed to generate two major peptides. GHRH is 42-44 amino acids, depending on the species, and is generally carboxyl-terminally amidated, except in rodents. A 30-amino acid GHRH-related peptide has been characterized from rat and mouse. Further processing to generate as many as five peptide forms has been reported (Nillni et al., 1999) although GHRH and GHRH-RP are the only well characterized products of the precursor. D, schematic structure of the secretin receptor. Shown is the predicted amino acid sequence and membrane topology of the human secretin receptor. Highlighted in white lettering on a black background are residues at the junctions of the exons, based on the genomic structure.

The long and structurally complex NH₂ terminus is critical for binding and action of the natural peptide agonist ligand (Holtmann et al., 1995). This is an important signature region of class II G protein-coupled receptors that contains six conserved cysteine residues believed to be involved in three intradomain disulfide bonds critical for establishing functional receptor conformation (Asmann et al., 2000). The secretin receptor NH₂ terminus also contains a cysteine residue in a nonconserved position that is not involved in a disulfide bond (Asmann et al., 2000). The functional importance of this region has been established by truncation, site-directed mutagenesis, chimeric receptors, and photoaffinity labeling studies (Dong et al., 1999a,b, 2000, 2002; Holtmann et al., 1995, 1996a; Park et al., 2000). It is remarkable that photolabile residues situated throughout the pharmacophoric domain of secretin, in positions 6, 22, and 26 have all been shown to covalently label residues within the NH₂ terminus of the secretin receptor (Dong et al., 2002). Extracellular loop domains of the secretin receptor have also been shown to be important regions for secretin binding, acting as important complements to the NH₂-terminal region (Holtmann et al., 1996a). The precise mechanism of peptide binding and the roles of each domain have not yet been well defined.

The classical sites for secretin receptor localization are on epithelial cells within the pancreatic and biliary ducts (Ulrich et al., 1998). These are the sites of stimulation of bicarbonate-rich fluid that is important to neutralize gastric acid emptied into the duodenum. Additionally, secretin receptors are present on pancreatic acinar cells, gastric epithelial cells, intestinal epithelial cells, Brunner's glands, gastric and intestinal smooth muscle cells, and certain areas of the brain. Receptors at these sites contribute to pancreatic exocrine secretion and growth, stimulation of gastric pepsinogen secretion, inhibition of gastric acid secretion, and inhibition of gastric emptying and intestinal motility. As noted above, these actions all work in concert to protect the upper intestinal tract from the major stimulants of secretin secretion, acidic chyme in the lumen. Additionally, there have been potential roles of secretin proposed for islet cell, renal, and cardiac function, although these actions are not well defined or understood.

Like most of the other receptors in the class II G protein-coupled receptor family, the secretin receptor has been shown to couple to G_s; the secretin receptor also couples to G_q. Receptor activation leads to increases in both cAMP and intracellular calcium (Trimble et al., 1987). Promiscuous coupling is typical of this receptor family and, like other members, the G_s coupling and cAMP signaling occur at the lowest concentrations of hormone and represents the physiological signaling pathway. The G_q coupling and intracellular calcium response occur in response to concentrations of secretin more than 100-fold higher than those stimulating the other pathway. Additional events along these signaling pathways have not been thoroughly examined or described.

The secretin receptor is phosphorylated in response to agonist action, although the specific functional impact of this biochemical event is not well established (Ozcelebi et al., 1995; Holtmann et al., 1996b). The kinases implicated in secretin receptor phosphorylation include G protein-coupled receptor kinases and protein kinase C (Ozcelebi et al., 1995; Holtmann et al., 1996b; Shetzline et al., 1998). There is no current information regarding action of G protein-coupled receptor phosphatases that might be regulated and act on this receptor (Lutz et al., 1993). The secretin receptor has also been demonstrated to be internalized into the cell in response to agonist occupation (Holtmann et al., 1996b). This has been best studied in model cellular systems, and nothing is yet known about the behavior of this receptor as it naturally resides on various cellular populations.

Recombinant secretin receptors expressed on Chinese hamster cell lines have been extremely useful for analysis of potential ligands and agonist activity (Ulrich et al., 1993). The rat pancreatic acinar cell is a natural site of secretin receptor expression that is a classical cell biological model system. A problem with its use is the coexpression of VPAC1 receptors that bind and respond to low concentrations of VIP and high concentrations of secretin. These have contributed to the older physiologic literature that describes secretin-preferring and VIP-preferring receptors being expressed on these cells (Gardner et al., 1976).

The major physiological roles for secretin relate to establishing and maintaining an optimal intraluminal milieu in the duodenum and upper jejunum for digestion to take place (Mutt, 1980). Once gastric acid enters the duodenum, it can damage the mucosal cells, precipitate bile acids, and inactivate pancreatic enzymes. Secretin is secreted in response to the acid load and many of its actions involve the direct stimulation of alkaline bicarbonate-rich fluid or the slowing of gastric emptying or intestinal transit to minimize acid exposure and to provide optimal opportunity for neutralization.

Secretin administration has been used clinically as a provocative test for gastrin-secreting islet cell tumors (Isenberg et al., 1972). Although the normal islet cell does not express the secretin receptor, gastrinoma cells often express this receptor (Chiba et al., 1989). As noted above, one of the established causes for a false-negative provocative test in the gastrinoma syndrome is the missplicing of the secretin receptor (Ding et al., 2002b).

	III. The Glucagon Receptor

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Glucagon is a 29-amino acid peptide (Bromer et al., 1956) originally isolated from a side fraction of purified insulin (Kenny, 1955) as a hyperglycemic factor originating from the pancreas (Kimball and Murlin, 1923). Its primary structure is identical in most mammals including man, although some amino acid sequence changes are noted in glucagons from guinea pig or nonmammalian vertebrates (Irwin, 2001). Glucagon is synthesized mainly in the A-cells present at the periphery of the islets of Langerhans (Baum et al., 1962) and is also detected in specific cells in the stomach and intestine in some species (Baetens et al., 1976), as well as in specialized neurons of the central nervous system. Isolation of cDNAs encoding glucagon (Lund et al., 1982; Lopez et al., 1983) showed that the peptide is produced from a 160-amino acid precursor, proglucagon, which also contains two additional glucagon-like sequences at its carboxyl terminus (GLP-1 and GLP-2), which were subsequently shown to display specific biological activities (Drucker, 1998).

Tissue-specific post-translational processing of the NH₂-terminal portion of proglucagon (Fig. 3A) (Mojsov et al., 1986; reviewed in Bataille, 1996b) leads to production of glucagon in the pancreatic A-cells. In the intestinal L-cells and central nervous system, the carboxyl-terminally extended forms, glicentin and oxyntomodulin (Bataille, 1996a), as well as GLP-1 and GLP-2 and two intervening peptides, IP-1 and IP-2 are produced. Further processing of glucagon may also produce the carboxyl-terminal undecapeptide miniglucagon, a powerful inhibitor of insulin secretion (Dalle et al., 1999, 2002).

The biological activities of glucagon are directed mostly toward opposing insulin action in the liver in the control of glucose metabolism primarily via stimulation of glycogenolysis (Sutherland, 1950; reviewed in Stalmans, 1983) and gluconeogenesis from lactate, pyruvate, glycerol, and certain amino acids (Claus et al., 1983). Similarly, the ratio of circulating levels of insulin and glucagon can shift lipid metabolism from storage to release in specific tissues such as the liver (Steinberg et al., 1959; Eaton, 1977). Glucagon is also able to directly stimulate insulin release (Samols et al., 1966) through its own receptors expressed on pancreatic -cells (Kawai et al., 1995; Moens et al., 1998). Although more likely pharmacological than physiological, glucagon administration produces positive inotropic and chronotropic effects on the heart (Farah, 1983), exerts spasmolytic effects on gastrointestinal smooth muscle (Diamant and Picazo, 1983) and growth hormone-releasing activities (Merimee, 1983).

Rodbell and coworkers (Rodbell et al., 1971a,b) established that the glucagon receptor is involved in the activation of adenylate cyclase and that intracellular signaling is achieved through GTP-binding heterotrimeric G proteins of the G_s type. Besides this universally accepted mode of action, which was later shown to be shared by many other peptides and neurotransmitters, glucagon may also, at very low doses, activate the phospholipase C/inositol phosphate pathway leading to Ca²⁺ release from intracellular stores in a physiological context. The signal transduction pathways activated by the glucagon receptor coupled to calcium release include stimulation of adenylate cyclase via G_s, and experiments using BHK fibroblasts implicate a role for both G_s- and G_i-activated pathways in the Ca²⁺ response to glucagon (Wakelam et al., 1986; Hansen et al., 1998). Using the affinity-labeling approach, the hepatic glucagon receptor was isolated as a 62-kDa polypeptide that contained four N-linked oligosaccharide chains (Iyengar and Herberg, 1984).

Cloning of the rat glucagon receptor cDNA was achieved via two parallel and simultaneous approaches: expression cloning (Jelinek et al., 1993) and polymerase chain reaction-based cloning (Svoboda et al., 1993). Cloning of the human (Lok et al., 1994; MacNeil et al., 1994; Menzel et al., 1994) and mouse receptor genes (Burcelin et al., 1995) followed. The human glucagon receptor gene is localized to chromosome 17, at 17q25 (Lok et al., 1994; Menzel et al., 1994). The rat and mouse glucagon receptors are 485-amino acid seven transmembrane domain proteins with four N-linked glycosylation sites and a sequence motif in the third intracellular loop (RLAR) known to be required for G protein activation. The human receptor is shorter, with 477 amino acids and contains a similar (RLAK) G protein-coupling motif as well as four N-linked glycosylation sites. Mouse and rat receptors are very similar (93% identity in amino acid sequence), whereas the human receptor is only 80% identical to the mouse receptor (Sivarajah et al., 2001). The glucagon receptor has a region (FQG-hydr-hydr-VAx-hydr-YCFxEVQ)hydr being a hydrophobic and x any amino acidthat is highly conserved in all the members of the glucagon/secretin receptor subfamily. A cDNA encoding an amphibian glucagon receptor was obtained from Rana tigrina rugulosa, the first nonmammalian glucagon receptor characterized (Ngan et al., 1999). More recently, the structures of the receptors from two more amphibians (Xenopus laevis and Rana pipiens) were determined (Sivarajah et al., 2001).

Glucagon receptor expression in islet cells is up-regulated by glucose, and glucose-responsive sequences have been identified in the glucagon receptor gene promoter (Abrahamsen and Nishimura, 1995; Yamato et al., 1997; Portois et al., 1999). Agents such as forskolin and 3-isobutyl-1-methylxanthine, which increase levels of cAMP, and the glucocorticoid dexamethasone, inhibit glucagon receptor expression in rat islets, whereas somatostatin, which reduces cAMP, increases levels of islet glucagon receptor RNA transcripts (Abrahamsen and Nishimura, 1995). Similarly, glucose increases and agents that augment levels of cAMP decrease glucagon receptor expression in primary cultures of rat hepatocytes (Abrahamsen et al., 1995). Furthermore, levels of hepatic glucagon receptor mRNA transcripts are down-regulated following exposure to glucagon in a dose-dependent manner (Abrahamsen et al., 1995).

Extensive analysis of glucagon receptor sequences has identified specific amino acids essential for ligand binding and signal transduction (Unson and Merrifield, 1994; Christophe, 1996; Buggy et al., 1997; Cypess et al., 1999; Unson et al., 2000). The importance of an aspartic acid residue in the extracellular domain for ligand binding was demonstrated (Carruthers et al., 1994). Structure-function studies (Unson et al., 1995) resulted in the conclusions that 1) all seven transmembrane helices are required for the proper folding and processing of the receptor; 2) glycosylation does not play an essential role in its membrane localization or its activity; 3) the amino-terminal extracellular portion is required for ligand binding; and 4) most of the distal carboxyl-terminal tail is not necessary either for ligand binding or for coupling to adenylate cyclase. The 206-219 segment of the first extracellular loop is important for both glucagon binding and receptor activation (Unson et al., 2002). Deletion of residues 252-259 corresponding to the second intracellular loop appears to lock the protein in the conformation promoted by divalent cations and prevents the protein from normal coupling to G_s, and the intracellular i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G protein-coupled receptors (Cypess et al., 1999). The human glucagon receptor has been shown to interact with receptor activity modifying proteins (RAMPs), specifically RAMP2, in transfected fibroblasts (Christopoulos et al., 2002); however, the potential biological significance of this interaction for glucagon receptor expression and activity in vivo has not yet been determined.

The distribution of the glucagon receptor was studied in the rat and found to be expressed mainly in liver and kidney and, to a lesser extent, in heart, adipose tissue, spleen, thymus, adrenal glands, pancreas, cerebral cortex, and throughout the gastrointestinal tract (Svoboda et al., 1994; Dunphy et al., 1998). Ligand binding studies have identified glucagon binding sites in rat kidney tubules including the thick ascending limb of Henle's loop, the distal convoluted tubule, and the collecting tubule (Butlen and Morel, 1985). In the rat brain, radioligand binding studies detected glucagon binding sites in the olfactory tubercle, hippocampus, anterior pituitary, amygdala, septum, medulla, thalamus, olfactory bulb, and hypothalamus (Hoosein and Gurd, 1984b).

No spontaneous mutations leading to constitutively active glucagon receptors have been found in humans. A missense mutation leading to a Gly⁴⁰ to Ser substitution has been associated with type 2 diabetes mellitus in French and Sardinian subjects (Hager et al., 1995) potentially associated with decreased glucagon-dependent insulin secretion. Alternatively, this mutation may be in linkage disequilibrium with another gene located in the same region, as another polymorphism found in intron 8 of the receptor cosegregates with the Gly⁴⁰Ser mutation in all individuals tested. A recent study indicates that there is no linkage between this mutation and type 2 diabetes in Brazilian patients (Shiota et al., 2002). It has also been suggested that this missense mutation may be associated, in some individuals, with essential hypertension (Morris and Chambers, 1996).

Authentic glucagon receptors, distinct from receptors for GLP-1 that may also recognize high concentrations of glucagon within the islet (Moens et al., 1998), have been detected in insulin-secreting -cells by several approaches (Kawai et al., 1995; Dalle et al., 1999; Huypens et al., 2000) including use of specific functional antagonists and identification of the mRNA encoding the glucagon receptor by Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) experiments. Islet glucagon receptors are coupled to adenylate cyclase and trigger insulin secretion. This observation is intriguing and paradoxical in that glucagon, which exhibits glycogenolytic and gluconeogenic effects in the setting of hypoglycemia, is also able to release insulin. Recent observations have demonstrated that miniglucagon, the carboxyl-terminal glucagon fragment also present in mature secretory granules of the A-cells, is released together with native glucagon (Dalle et al., 2002). Because of the huge difference in affinity between the two peptides for their respective receptors (3 to 4 orders of magnitude higher for the smaller peptide), miniglucagon, a very efficient inhibitor of insulin release that acts by closure of the -cell voltage-gated calcium channels consecutive to membrane repolarization (Dalle et al., 1999), completely blocks any possible insulinotropic effect of glucagon. Consistent with these findings, although exogenous glucagon stimulates insulin secretion, endogenously released glucagon has no effect on the magnitude of glucose-induced insulin secretion (Moens et al., 2002).

Peptide antagonists of the glucagon receptor have been described, most of which lacked the amino-terminal histidine residue and contained a modified amino acid at position 9 such as des-His¹-[Nle⁹-Ala¹¹-Ala¹⁶] glucagon (Unson et al., 1991, 1993). A nonpeptidic competitive antagonist of the glucagon receptor (NNC 92-1687) has also been described (Madsen et al., 1998). There remains active interest in the search for nonpeptide glucagon receptor antagonists, which may be useful for the treatment of type 2 diabetes (Ling et al., 2001a, 2002; Petersen and Sullivan, 2001; Ladouceur et al., 2002).

Mice with a targeted disruption of the glucagon receptor, leading to a complete ineffectiveness of glucagon on its target tissues, have  cell hyperplasia near normal levels of insulin, mild fasting hypoglycemia, normal levels of fasting cholesterol and triglycerides, and improved glucose tolerance despite very high levels of circulating glucagon (Parker et al., 2002; Gelling et al., 2003).

Nothing is known about the putative molecular structure of receptors for the related proglucagon-derived peptides oxyntomodulin (glucagon plus a carboxyl-terminal extension), glicentin (glucagon plus both an NH₂- and carboxyl-terminal extension), or miniglucagon (the carboxyl-terminal [19-29] glucagon sequence) (Fig. 3). Whether these peptides act through known members of the secretin receptor family or exert their actions through distinct novel receptors, remains unclear. However, it must be noted that

1.

Oxyntomodulin and glicentin, secreted from the same intestinal L-cells as GLP-1, display specific biological activities directed toward regulation of gastric acid secretion and gut motility (Bataille, 1996a), and these peptides appear to act via receptors linked to both the inositol phosphate and cyclic AMP pathways (Rodier et al., 1999). Intracerebroventricular administration of oxyntomodulin inhibits food intake in rats, and this effect is blocked by the GLP-1 receptor antagonist exendin-(9-39); however, the specific receptor that mediates this effect has not yet been identified (Dakin et al., 2001). Administration of glicentin to rats or mice produces small bowel growth, although not to the same extent as the more potent intestinotrophic peptide GLP-2 (Drucker et al., 1996).

2.

Miniglucagon, produced from circulating glucagon in target-tissues (Bataille, 1996b) and present in glucagon-containing secretory granules (Dalle et al., 2002), activates receptors coupled to the plasma membrane calcium pump (Mallat et al., 1987) via a Gs or Gs-like GTP-binding protein (Lotersztajn et al., 1990) in liver and to potassium channels via a Gi or Go GTP-binding protein in pancreatic -cells (Dalle et al., 1999). Studies examining the biological actions of glicentin, oxyntomodulin, or miniglucagon in mice with targeted disruption of the related glucagon or GLP-1 receptors have not yet been reported.

	IV. The Glucagon-Like Peptide-1 Receptor

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Peptidergic signals derived from the intestine augment the insulin response induced by nutrients ("the incretin effect") (Dupre and Beck, 1966; Fehmann et al., 1995a). This functional connection between the intestine and the islets of Langerhans was termed the "incretin axis" or "entero-insular-axis" (Unger and Eisentraut, 1969; Fehmann et al., 1995a). The gut-derived peptides GLP-1 and GIP are important mediators in this axis (Fehmann et al., 1995a; Drucker, 1998; Kieffer and Habener, 1999).

GLP-1, together with GLP-2, oxyntomodulin, and glicentin, is derived from post-translational processing of proglucagon in the intestinal L-cells of the small and large intestine (Mojsov et al., 1986) (Fig. 3). GLP-1 stimulates insulin secretion in a glucose-dependent manner via a specific receptor expressed on islet -cells. GLP-1 also increases proinsulin gene transcription and insulin production and suppresses glucagon secretion from islet -cells (Fehmann and Habener, 1992; Fehmann et al., 1995a; Drucker, 1998). Whether the effect of GLP-1 on inhibition of glucagon secretion is direct, via -cell expression of the GLP-1 receptor, or indirect, perhaps via stimulation of insulin or somatostatin secretion, remains unclear (Moens et al., 1996; Heller et al., 1997). GLP-1 has central nervous system effects resulting in delayed gastric emptying (Schirra et al., 1997) and appetite regulation (Turton et al., 1996; Gutzwiller et al., 1999; Verdich et al., 2001), and the circulating peptide may gain access to the brain from the periphery by simple diffusion (Kastin et al., 2002). Systemic administration of GLP-1 in rodents activates the sympathetic nervous system leading to increased tyrosine hydroxylase gene transcription, enhanced sympathetic outflow, and increased heart rate and blood pressure (Barragan et al., 1999; Yamamoto et al., 2002).

GLP-1 receptors were first identified by a combination of radioligand binding experiments and measurements of cyclic AMP accumulation using rat insulinoma-derived RIN1046-38 cells (Drucker et al., 1987; Goke and Conlon, 1988) followed by localization on additional rodent insulinoma cell lines (Fehmann et al., 1995a) as well as on rat (Moens et al., 1996) and human (Fehmann et al., 1995b) pancreatic islet -cells and somatostatin-secreting cells (Fehmann and Habener, 1991; Gros et al., 1992). GLP-1 binding sites have also been identified on isolated rat gastric parietal cells (Uttenthal and Blazquez, 1990; Schmidtler et al., 1994), human gastric cancer cells (HGT-1) (Hansen et al., 1988), solubilized membranes of rat epididymal adipose tissue (Valverde et al., 1993), 3T3-L1 adipocytes (Montrose-Rafizadeh et al., 1997), membranes from the rodent thyrotrope cell line -TSH (Beak et al., 1996), and in rat lung (Richter et al., 1990, 1991) and brain (Shimizu et al., 1987; Uttenthal and Blazquez, 1990; Goke et al., 1995; Wei and Mojsov, 1995).

Analysis of data obtained from binding experiments with RINm5F cells revealed that GLP-1 binds to a single class of binding sites (Goke and Conlon, 1988). Cross-linking studies with ¹²⁵I-GLP-1 demonstrate a single band with an apparent molecular mass of 63,000 (Goke et al., 1989a, 1992). The GLP-1 receptor protein is glycosylated and glycosylation may modulate receptor function (Goke et al., 1994). Agonists at the receptor include GLP-1(7-37), GLP-1(7-36)amide, the Heloderma suspectum peptides exendin-3 and exendin-4 (Goke et al., 1993; Thorens et al., 1993), and labeled ligands such as fluorescein-Trp25-exendin-4 (Chicchi et al., 1997) ¹²⁵I-GLP-1, and Tyr39-exendin-4. In contrast, structurally related members of the glucagon family such as GLP-2, glucagon, and GIP do not exhibit cross-reactivity at the GLP-1 receptor at physiologically relevant concentrations.

Agonist potencies at the receptor exhibit a K_d of 0.3 nM for GLP-1(7-37)/(7-36)amide and a K_d of 0.1 nM for the naturally occurring Gila monster peptide exendin-4 (Goke et al., 1993). The truncated lizard peptide GLP-1 receptor antagonist exendin-(9-39) (Goke et al., 1993; Thorens et al., 1993) exhibits a K_d of 2.9 nM (Goke et al., 1993; Thorens et al., 1993). This compound has been successfully utilized for in vitro (Goke et al., 1993; Thorens et al., 1993) and in vivo studies (Kolligs et al., 1995; Schirra et al., 1998) for elucidation of the physiological importance of the GLP-1 receptor. A small nonpeptide ligand (T-0632) for the GLP-1 receptor has been described that binds to the amino-terminal hormone binding domain with micromolar affinity (Tibaduiza et al., 2001). Interestingly, this (non)peptide antagonist exhibits ~100-fold selectivity for the human versus the highly homologous rat GLP-1 receptor, due to the presence of Trp versus Ser at position 33 in the human versus rat receptors, respectively (Tibaduiza et al., 2001). Hence it may be feasible to develop nonpeptide orally bioavailable small molecule GLP-1 receptor modulators for therapeutic purposes.

Molecular characterization of the GLP-1 receptor was achieved by cloning the rat and human -cell GLP-1 receptor cDNAs (Thorens, 1992; Dillon et al., 1993; Graziano et al., 1993; Thorens et al., 1993; Van Eyll et al., 1994) followed by isolation of cDNAs encoding the rat lung and the brain GLP-1 receptor (Lankat-Buttgereit et al., 1994; Wei and Mojsov, 1995). The human receptor protein consists of 463 amino acids (Van Eyll et al., 1994). The rat and human GLP-1 receptors exhibit 90% sequence identity at the amino acid level. The receptor sequence contains a large hydrophilic, extracellular domain preceded by a short leader sequence required for receptor translocation across the endoplasmic reticulum during biosynthesis, and seven hydrophobic membrane-spanning domains that are linked by hydrophilic intra- and extracellular loops (Thorens and Widmann, 1996). Distinct amino acids within the amino-terminal domain of the receptor are crucial for ligand binding (Parker et al., 1998; Tibaduiza et al., 2001; Wilmen et al., 1997), and the region encompassing transmembrane domains 1 to 3 is also involved in ligand binding (Xiao et al., 2000b). Different domains in the third intracellular loop of the GLP-1 receptor are responsible for specific G protein-coupling (and G_s, G_i, and G_o activation) (Hallbrink et al., 2001), and in the best studied cellular model, islet -cells, GLP-1 receptor signaling acts predominantly via G_s to increase cAMP accumulation; however, activation of downstream signaling pathways may occur in a protein kinase A-independent manner (Bode et al., 1999; Holz et al., 1999). GLP-1-mediated closure of ATP-sensitive potassium (K_ATP) channels, and the differential effect of ADP levels on K_ATP channel closure may provide a cellular mechanism for the glucose-sensitivity of GLP-1 action in -cells (Light et al., 2002).

The human GLP-1 receptor gene was localized to the long arm of chromosome 6 (hchr 6p21) (Stoffel et al., 1993). The GLP-1 receptor gene spans 40 kb and consists of at least 7 exons. The 5'-flanking and promoter region of the human GLP-1 receptor gene has been cloned and functionally characterized (Lankat-Buttgereit and Goke, 1997). The cell- and tissue-specific transcriptional regulation of GLP-1 receptor expression has been studied in cell transfection experiments and is mainly achieved by selective utilization of positive and negative control sequences and silencing elements, the latter located between 574 and 2921 (Galehshahi et al., 1998; Wildhage et al., 1999).

Studies investigating the distribution of rat and human GLP-1 receptor mRNA by RNase protection and RT-PCR detected GLP-1 receptor mRNA transcripts in pancreatic islets, lung, brain, stomach, heart, and kidney but not in liver, skeletal muscle or adipose tissue of most species (Wei and Mojsov, 1995; Bullock et al., 1996). In contrast, GLP-1 receptor transcripts have been identified in canine muscle and adipose tissue (Sandhu et al., 1999). Although quantitative comparative analyses of the levels of GLP-1 receptor expression in distinct isolated cell types are not yet available, Northern blot and RNase protection analyses demonstrates comparatively greater levels of GLP-1 receptor mRNA transcripts in heart and lung compared with other tissues (Thorens, 1992; Wei and Mojsov, 1995; Bullock et al., 1996). In rat brain, GLP-1 receptors have been found in the lateral septum, subfornical organ, thalamus, hypothalamus, interpeduncular nucleus, posterodorsal tegmental nucleus, area postrema, inferior olive, and nucleus of the solitary tract (Goke et al., 1995; Shughrue et al., 1996). Activation of brain GLP-1 receptors likely occurs via GLP-1 produced in the brainstem, which then is transported to distant regions of the central nervous system (Drucker and Asa, 1988; Jin et al., 1988; Larsen et al., 1997; Merchenthaler et al., 1999) and via activation of GLP-1 receptors in the area postrema that then activate brainstem GLP-1+ neurons (Kastin et al., 2002; Yamamoto et al., 2002).

The GLP-1 receptor is functionally coupled to adenylate cyclase (Drucker et al., 1987) via the stimulatory G protein G_s. GLP-1-binding at pancreatic -cells increases free cytosolic calcium concentrations after cell depolarization in some but not all cell types (Goke et al., 1989b; Lu et al., 1993; Yada et al., 1993; Holz et al., 1995, 1999; Bode et al., 1999). GLP-1-dependent stimulation of intracellular calcium may occur via a ryanodine-sensitive pathway (Holz et al., 1999), and in a cAMP-dependent, protein kinase A-independent manner through small G proteins distinct from G_s (Kang et al., 2001; Kashima et al., 2001).

GLP-1 receptor function has been studied using various approaches. Radioligand assays were used to characterize binding at the endogenous receptor expressed in rat insulinoma cell lines (Goke and Conlon, 1988; Fehmann et al., 1995a), recombinant receptors expressed in transfected COS cells (Thorens, 1992; Thorens et al., 1993) or transfected Chinese hamster lung fibroblast (rCHL) cells (Van Eyll et al., 1994). Homologous desensitization and internalization of the GLP-1 receptor is strictly dependent on the phosphorylation of three serine doublets within the cytoplasmic tail (Widmann et al., 1997). Experiments with mutant GLP-1 receptors revealed that the number of phosphorylation sites correlated with the extent of desensitization and internalization. However, the two processes showed a different quantitative impairment in single versus double mutants suggesting different molecular mechanisms controlling desensitization and internalization (Widmann et al., 1997). The specific identity of the protein kinases regulating GLP-1 receptor phosphorylation and receptor desensitization remain unclear.

Glp1r^/ mice with a targeted genetic disruption of the GLP-1 receptor gene demonstrate modest glucose intolerance and fasting hyperglycemia with defective glucose-stimulated insulin secretion (Scrocchi et al., 1996). Glp1r^/ mice also exhibit subtle abnormalities in the hypothalamic-pituitary-adrenal axis, specifically, an abnormal corticosterone response to stress (MacLusky et al., 2000). Despite the putative importance of GLP-1 as a satiety factor, even combined disruption of leptin and GLP-1 action as exemplified by generation and analysis of a double mutant ob/ob:Glp1r^/ mouse, did not modify weight gain or feeding behavior beyond that observed in the control ob/ob mouse alone (Scrocchi et al., 2000). Similarly, although exogenous administration of GLP-1 receptor ligands stimulates islet neogenesis and proliferation (Stoffers et al., 2000), complete disruption of Glp1r^/ signaling produces only modest defects in islet formation and topography (Ling et al., 2001b) and does not impair up-regulation of insulin gene expression or development of islet hyperplasia in the setting of leptin deficiency (Scrocchi et al., 2000). These findings illustrate the complexity of inferring the physiological importance of receptor function from studies of knockout mice in vivo (Seeley et al., 2000).

GLP-1 receptor agonists are being evaluated for clinical use as antidiabetic agents (Byrne and Goke, 1996; Drucker, 2001a; Zander et al., 2002). Since the half-life of the naturally occurring peptide in plasma is too short for optimal clinical use, long-acting degradation resistant GLP-1 analogs have now been developed (Deacon et al., 1998; Ritzel et al., 1998; Burcelin et al., 1999a; Siegel et al., 1999; Doyle et al., 2001; Xiao et al., 2001), and these analogs, together with the lizard peptide exendin-4, are being assessed in studies of patients with type 2 diabetes (Agerso et al., 2002; Egan et al., 2002; Juhl et al., 2002).

The overexpression of the GLP-1 receptor in insulin-releasing INS-1 cells increases the potency and efficacy of D-glucose on insulin gene transcription by a putative autocrine signaling mechanism (Chepurny and Holz, 2002). This observation affirms the idea that -cell lines could be engineered for efficient glucose-dependent insulin synthesis and secretion by overexpression of the GLP-1 receptor. Alternatively, genetic engineering of cells for expression of GLP-1 receptor ligands has also been proposed (Burcelin et al., 1999b).

Activation of GLP-1 receptor signaling has been proposed as a therapeutic strategy for treatment of peripheral diabetic neuropathy and other neurodegenerative processes. GLP-1, and its longer-acting analog exendin-4, completely protected cultured rat hippocampal neurons against glutamate-induced apoptosis (Perry et al., 2002a), and GLP-1 promotes nerve growth factor-mediated differentiation in PC12 cells (Perry et al., 2002b). Activation of GLP-1 receptor signaling also promotes proliferative and anti-apoptotic actions in the endocrine pancreas, providing a potential opportunity for interventions directed at expanding -cell mass in subjects with diabetes (Li et al., 2003; Drucker, 2003).

	V. The Glucagon-Like Peptide-2 Receptor

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GLP-2 was first identified as a novel peptide encoded within the mammalian proglucagon cDNA sequence (Fig. 3) carboxyl-terminal to GLP-1 (Bell et al., 1983a,b), and subsequent isolation and characterization of the peptide from porcine and human small bowel confirmed the synthesis and liberation of full-length GLP-2(1-33) (Buhl et al., 1988). The GLP-2 amino acid sequence is flanked by pairs of dibasic residues characteristic of prohormone cleavage sites. GLP-2 is cosecreted along with GLP-1, oxyntomodulin, and glicentin from intestinal endocrine cells (Mojsov et al., 1986; Orskov et al., 1986). The principal role of GLP-2 appears to be the maintenance of growth and absorptive function of the intestinal mucosal villus epithelium (Drucker et al., 1996). GLP-2 administration to rodents enhances villus growth and increases small bowel mass, with weaker but detectable trophic effects observed in the large bowel and stomach (Drucker et al., 1997a,b; Tsai et al., 1997a,b). GLP-2 also rapidly up-regulates hexose transport and nutrient absorption (Cheeseman and Tsang, 1996; Brubaker et al., 1997) and enhances sugar absorption and intestinal adaptation in rats following major small bowel resection (Scott et al., 1998). GLP-2 reduces intestinal permeability in rodents within hours of peptide administration in vivo but has no effect on mucosal permeability when administered in vitro (Benjamin et al., 2000), consistent with the established indirect actions of GLP-2 (Drucker, 2001b).

The trophic and proabsorptive actions of GLP-2 have prompted studies of whether pharmacological GLP-2 administration may produce beneficial effects in rodent models of intestinal disease. GLP-2 treatment ameliorates the severity of small bowel enteritis and facilitates adaptive small bowel mucosal repair following surgical or chemical injury in both rats and mice (Chance et al., 1997; Scott et al., 1998; Boushey et al., 1999; Alavi et al., 2000; Prasad et al., 2000, 2001). A GLP-2 analog also reduces weight loss and facilitates mucosal healing in mice with experimental colitis (Drucker et al., 1999b). A pilot study of GLP-2 administration for 4 weeks to human subjects with short bowel syndrome produced significant improvement in lean body mass, intestinal histology, and energy retention (Jeppesen et al., 2001). Although the CNS actions of GLP-2 remain poorly understood, pharmacological administration of intracerebroventricular GLP-2 modestly and transiently reduces food intake in rats and mice (Tang-Christensen et al., 2000; Lovshin et al., 2001).

Although GLP-2 binding sites have not yet been reported on cell lines expressing an endogenous GLP-2 receptor, GLP-2 activates adenylate cyclase in rat hypothalamic and pituitary membranes (Hoosein and Gurd, 1984a), and administration of radiolabeled GLP-2 to rats results in detectable radioligand binding to intestinal epithelial cells along the crypt to villus axis (Thulesen et al., 2000). A cDNA encoding a GLP-2 receptor was isolated from hypothalamic and intestinal cDNA libraries using a combined PCR expression cloning approach (Munroe et al., 1999). The GLP-2 receptor cDNA encodes an open reading frame of 550 amino acids that gives rise to a structurally related member of the class 2 glucagon-secretin receptor family (Munroe et al., 1999). The GLP-2 receptor gene was localized to human chromosome 17p13.3 and has not yet been linked to specific human diseases. The GLP-2 receptor is expressed in a tissue-specific manner in the stomach, small and large intestine, central nervous system, and lung (Munroe et al., 1999; Yusta et al., 2000b). GLP-2 receptor expression in the human gut epithelium has been localized by immunohistochemistry to subsets of enteroendocrine cells in the stomach, and both small and large intestine using a polyclonal antiserum (Yusta et al., 2000b). Although the majority of human enteroendocrine cells do not express the GLP-2 receptor, all GLP-2 receptor-immunoreactive cells identified to date express one or more gut endocrine markers, including GIP, peptide YY, serotonin, chromogranin, and GLP-1 (Yusta et al., 2000b). In contrast, the same antisera did not identify GLP-2 receptor-immunopositive endocrine cells in rodents, where GLP-2 receptor RNA transcripts have been localized by in situ hybridization to subsets of enteric neurons (Bjerknes and Cheng, 2001). These findings imply an indirect model for GLP-2 action whereby GLP-2 released from enteroendocrine L-cells or rodent neurons stimulates the release of downstream mediators of GLP-2 action (Fig. 4). The downstream mediators are responsible for the proliferative, anti-apoptotic, and pro-absorptive effects of GLP-2; however, the identity of these GLP-2-dependent factors has not yet been established.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Schematic representation of GLP-2 action in the gastrointestinal epithelium via GLP-2 receptors expressed in the enteric nervous system (mouse) or enteroendocrine cells (human).

Consistent with the original description of GLP-2 action in the brain (stimulation of adenylate cyclase activity) in hypothalamic and pituitary membranes (Hoosein and Gurd, 1984a), GLP-2 increases intracellular cAMP in fibroblasts transfected with the rat or human GLP-2 receptor cDNA with an EC₅₀ of 0.58 nM, and binding studies demonstrate a K_d of 0.57 nM (Munroe et al., 1999; Yusta et al., 1999; DaCambra et al., 2000). In contrast, structurally related members of the glucagon peptide family such as glucagon, GLP-1, GIP, secretin, growth hormone-releasing factor, pituitary adenylate cyclase-activating peptide (PACAP), and VIP do not activate the transfected rat or human GLP-2 receptor at concentrations of 10 nM in vitro (Munroe et al., 1999; DaCambra et al., 2000). Structure-function analyses of GLP-2 ligand-receptor interactions demonstrate that both GLP-2(1-33) and GLP-2(1-34) are biologically active, and the ability of amino-terminally truncated or carboxyl-terminally extended GLP-2 derivatives to stimulate GLP-2 receptor-dependent cAMP accumulation in vitro correlates with the intestinotrophic properties of these peptides in a murine bioassay in vivo (Munroe et al., 1999).

The structure-function relationships for GLP-2 receptor activation have been examined through a combination of alanine scanning and position 2 substitution experiments using the human GLP-2 peptide sequence as a starting peptide and baby hamster kidney fibroblasts transfected with the rat GLP-2 receptor (BHK-GLP-2R cells) (DaCambra et al., 2000). The majority of position 2 h[GLP-2] substitutions exhibit normal to enhanced GLP-2R binding; in contrast, position 2 substitutions were less well tolerated for receptor activation as only Gly, Ile, Pro, -aminobutyric acid, D-Ala, or nor-Val substitutions enhanced GLP-2 receptor activation (DaCambra et al., 2000). Alanine-scanning mutational analyses revealed that alanine replacement at positions 5, 6, 17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding (DaCambra et al., 2000). Position 2 residue substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21, which all exhibited normal to enhanced GLP-2 receptor binding but g