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Endocrinology Vol. 143, No. 12 4646-4654
Copyright © 2002 by The Endocrine Society

ARTICLE

Isolation and Structure-Function Studies of a Glucagon-Like Peptide 1 Receptor from Goldfish Carassius auratus: Identification of Three Charged Residues in Extracellular Domains Critical for Receptor Function

Chung-Man Yeung, Svetlana Mojsov, Pui-Yee Mok and Billy K. C. Chow

Department of Zoology (C.-M.Y., P.-Y.M., B.K.C.C.), The University of Hong Kong, Pokfulam Road, Hong Kong, China; and Laboratory of Cellular Physiology and Immunology (S.M.), Rockefeller University, New York 10021-6399

Address all correspondence and requests for reprints to: Billy K. C. Chow, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong. E-mail: bkcc{at}hkusua.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A better understanding of the molecular mechanism of ligand-receptor interaction of glucagon-like peptide 1 (GLP-1) receptors (GLP-1Rs) is useful for the design of potent GLP-1 analogs that could potentially be used as a treatment for diabetic patients. Changes in the ligand and receptor sequences during evolution provide invaluable clues to evaluate the functional motifs of the receptor that are responsible for ligand interaction. For these reasons, in the present study, we have isolated and functionally characterized a GLP-1R from goldfish. Its amino acid sequence shows 50.8% and 52.3% identity with the human glucagon (hGLU) and GLP-1Rs, respectively, and 84.1% with the zebrafish GLP-1R (the only other GLP-1R isolated from teleost fish). Peptides that are structurally different from goldfish (gf)GLP-1, such as gfGLU and hGLU and human GLP-1 (7–36)amide, are also capable of stimulating this receptor, albeit with lower potencies than gfGLP-1. gfGLP-1 stimulates the formation of cAMP through the recombinant gfGLP-1R with EC50 = 0.18 nM, whereas EC50 values for gfGLU, human GLP-1 (7–36)amide, and hGLU are 0.53 nM, 0.9 nM, and 1.2 nM, respectively. These results indicate that the gfGLP-1R is structurally more flexible than its mammalian counterpart and that its binding pocket can accommodate a wider spectrum of peptide ligands. Previous studies demonstrated that the charged residues in the extracellular domains of mammalian GLP-1R, particularly those found in the N-terminal domain and the first exoloop, are important for ligand binding. We investigated the roles of the conserved charged residues in the function of the gfGLP-1R. Eleven mutant receptors were constructed, and the effects of mutations were determined by functional assays. Our results demonstrated that three charged residues (D113, R197, and D205) present in the extracellular domains are critical for receptor function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON-LIKE PEPTIDE 1 (GLP-1), one of the proglucagon-derived peptides, has important physiological functions in vertebrates. In mammals, some of its actions include stimulation of glucose-dependent insulin secretion, inhibition of glucagon secretion and gastric emptying, and (possibly) promotion of glycogenesis (1, 2, 3). The action of GLP-1 is mediated via a specific cell surface receptor, which has been cloned and characterized in rat (4) and human (5, 6, 7). The GLP-1 receptor (GLP-1R) belongs to a large family of seven membrane-spanning G-protein-coupled receptors (GPCRs), which also includes receptors for secretin (8), calcitonin (9), GHRH (10), glucose-dependent insulinotropic polypeptide (GIP) (11), and glucagon (12). These GPCRs activate adenylyl cyclase for signal transduction. Mechanisms involving calcium for signaling have also been reported for the GLP-1 receptor (13, 14, 15). The GLP-1 receptor is widely distributed in tissues, including brain, pancreas, intestine, lung, stomach, heart, and kidney (16, 17).

In contrast to mammalian GLP-1, fish GLP-1 has a glucagon-like activity, which opposes the actions of insulin (18). Apparently, there is a functional switch of GLP-1 from having glucagon-like activities in fish to being an insulinotropic secretagogue in mammals. Despite the functional differences of fish and mammalian GLP-1s, fish GLP-1 is insulinotropic in mammals (19), and mammalian GLP-1 has glucagon-like action in fish (20). Thus, fish and mammalian GLP-1s seem to be interchangeable in their functions. These early findings indicated that a putative GLP-1 receptor should be present in fish and that the fish GLP-1 receptor should be able to interact with the mammalian GLP-1 peptide. This hypothesis was confirmed when the zebrafish (zf)GLP-1R was cloned and found to bind with similar affinity to the zfGLP-1 and mammalian GLP-1 (7–36)amide (2). In addition, there was evidence suggesting that fish glucagon [goldfish (gf)GLU] and GLP-1, though having similar biological function, act via different receptors (21). We have previously characterized a proglucagon cDNA from goldfish Carassius auratus, which encodes both glucagon and GLP-1 sequences (22). In the present study, we have isolated and functionally characterized a GLP-1 receptor from goldfish, with a goal to understand the molecular mechanisms governing the interaction of GLP-1 receptors both with its specific ligands and with the intracellular G proteins. This understanding is essential for future design of potent GLP-1 analogs for the treatment of diabetes mellitus. Several studies have been performed previously and have identified the structural features in the sequence of the mammalian GLP-1 receptors that play a role in the interaction with the ligand (23, 24) and in signal transduction (25, 26, 27, 28). They have demonstrated that the extracellular domains, especially the large N-terminal (NT) domain and the first exoloop, are important for ligand binding. To gain insights into the role of particular amino acid residues in the function of the gfGLP-1R, we constructed and characterized, in the present study, 11 receptor mutants. These mutations were located within the putative ligand-binding domains of the gfGLP-1R. The mutants were designed by comparing the gfGLP-1R sequence with the conserved regions of various mammalian GLP-1 receptors. Functional characterization of the mutants indicated that 3 charged residues (at positions 113, 197, and 205) are critical for function of the gfGLP-1R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of the gfGLP-1R
Using the first-strand cDNA prepared from the goldfish brain mRNA as the PCR template, a partial cDNA clone corresponding to the transmembrane domains (TMDs) 2–6 of a putative gfGLP-1R was obtained by a two-step PCR approach (29, 30). The partial cDNA clone was then used as a probe to screen a goldfish brain and pituitary cDNA library, which was used previously for the isolation of goldfish VPAC1, PAC1, and peptide histidine isoleucine/peptide histidine valine receptors (30, 31, 32). A full-length cDNA clone encoding the gfGLP-1R was isolated and excised to a phagemid pBK-CMV-gfGLP-1R according to instructions from the manufacturer (Stratagene, Cambridge, UK). The clone was sequenced from both strands using a T7 sequencing kit (Amersham Pharmacia Biotech, Arlington Heights, IL) by synthetic primers and by subcloning of restriction fragments. The DNA sequences were analyzed by DNasis (Hitachi Scientific Instruments, Inc., San Bruno, CA) and GeneWorks (Intelligenetics, Inc., Mountain View, CA).

Functional expression of the gfGLP-1R in COS-7 cells
To remove the 5' end of the lac Z gene from the vector pBK-CMV that could potentially affect expression in eukaryotic cell lines, a Spe I/Nhe I restriction fragment was released from the clone pBK-CMV-gfGLP-1R. The receptor construct was functionally expressed in COS-7 cells for the cAMP assays as described earlier (29, 30). Briefly, 0.2 million cells per well were seeded onto 6-well plates (Costar, San Diego, CA) 2 d before the assays. The cells were washed once with MEM/BSA (BSA, 1 mg/ml) and incubated with the same medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (Sigma, St. Louis, MO) for 30 min at 37 C. Peptides were added to stimulate the cells for 45 min. After stimulation, the cells were lysed by 1 ml cold ethanol. The cell debris was discarded after centrifugation (14,000 rpm for 10 min), and the supernatant was dried for cAMP quantification using an RIA kit (NEN Life Science Products, Boston, MA). Human peptides were purchased from Bachem California, Inc. (Torrance, CA), and goldfish peptides were synthesized by Peninsula Laboratories, Inc. (Belmont, CA) according to the deduced amino acid sequences of goldfish proglucagon cDNA (22).

Tissue distribution of the gfGLP-1R
Total RNA from various tissues, including brain, gall bladder, gill, male gonad, female gonad, lower intestine, upper intestine, kidney, liver, heart, pituitary, muscle, and spleen, were prepared using the acid guanidinium thiocyanate-phenol-chloroform extraction method (33). Messenger RNA was isolated by the PolyATtract mRNA isolation system (Promega Corp., Madison, WI), and first-strand cDNA was synthesized using 1 µg mRNA from each tissue (22). RT-PCR was performed using a pair of gene-specific primers (5'-CCC GGA GCT GGC GGC CTC GGC CTC-3' and 5'-ATG GCA CTC CTG CAA TGT GTG TGG-3') to detect the tissue distribution of the gfGLP-1R. The expected size of the amplified fragment was 1135 bp. The reaction conditions were 40 sec at 94 C, 1 min 30 sec at 68 C, and 1 min 30 sec at 72 C, respectively, for 30 cycles. The PCR products were analyzed on a 1% agarose gel and transferred to Hybond-N membrane (Amersham Pharmacia Biotech) for subsequent hybridization using [{alpha}-32P]-deoxycytidine triphosphate (Amersham Pharmacia Biotech)-labeled cDNA probe. In addition, another pair of PCR primers (5'-CAC TGT GCC CAT CTA CGA G-3' and 5'-CCA TCT CCT GCT CGA AGT C-3') was used to amplify the goldfish ß-actin cDNA (22) as a control (with an expected product size of 200 bp). To detect the expression pattern of the receptor in the brain, the tissue was dissected into eight parts, including olfactory bulbs and tracts, telencephalon, hypothalamus, optic tectum-thalamus, cerebellum, medulla, spinal cord, and pituitary, as previously described (34). RT-PCR analysis was performed using the first-strand cDNA synthesized from each part, as mentioned earlier.

Construction of the gfGLP-1R mutants
The receptor cDNA was released from pBK-CMV-gfGLP-1R by Sma I and BamH I digestion and cloned into the pALTER-1 vector (Promega Corp.). Single-stranded DNA, generated from the phagemid, was used as the template for mutagenesis reactions, using the Altered Sites II in vitro Mutagenesis System (Promega Corp.). Mutagenic primers were purchased from Life Technologies, Inc. (Gaithersburg, MD)and 5'-phosphorylated as recommended by Promega Corp. The sequences of these primers are listed in Table 1Go. Mutants were sequenced to confirm their identities and cloned back to the pBK-CMV expression vector for subsequent functional studies.


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  		Table 1. The sequences of mutagenic primers used in the present study

 
Functional studies of the gfGLP-1R mutants in Chinese hamster ovary (CHO) cells
The wild-type (wt) and mutant receptors were functionally expressed in CHO cells by transient transfection using Lipofectamine reagent (Life Technologies, Inc.). The effects of mutations on the receptor were determined by cAMP assays. In summary, 0.2 million CHO cells per well were seeded onto 6-well plates (Costar) and incubated at 37 C for 48 h. For each well, 1 µg of the receptor construct was cotransfected with 0.25 µg pCMV·SPORT-ßgal plasmid DNA (Life Technologies, Inc.) for 6 h using 4 µl Lipofectamine reagent (Life Technologies, Inc.). Cell lysate was prepared at 48 h, for measuring the ß-galactosidase activity by a ß-galactosidase enzyme assay system (Promega Corp.) to monitor the efficiency of transfection. The cells were stimulated with different peptides (at 10 nM) to evaluate the effects of mutations on the receptor cAMP responses, and cAMP assays were performed as mentioned earlier. Three independent experiments (n = 3), each in triplicate, were performed. Data were normalized by the ß-galactosidase activities and expressed as percent of wt (mean ± SEM). Results were analyzed using GraphPad Software, Inc. Prism by one-way ANOVA with Dunnett’s post test (GraphPad Software, Inc., San Diego, CA). Differences were considered significant when P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of the gfGLP-1R
We have isolated a GLP-1 receptor from the goldfish. The receptor cDNA is 3671 bp in length, containing a single open reading frame of 1536 bp, which predicts to encode a protein of 512 amino acids (Fig. 1Go. A Kyte-Doolittle hydrophobicity analysis of the deduced protein sequence (data not shown) indicated that the protein contains seven segments of hydrophobic residues characteristic for all GPCRs. Phylogenetic analysis also revealed that the receptor is closely related to other GLP-1 receptors in the glucagon/secretin receptor family (data not shown), and it shares 50.8% and 52.3% of amino acid sequence identities with human glucagon (hGLU) and GLP-1 receptors, respectively. As expected, the gfGLP-1R is the most closely related to the zfGLP-1R (84.1% identity). There are three potential N-linked glycosylation sites (Asn-X-Ser/Thr) present at positions 54, 73, and 106, and nine conserved cysteine residues located at positions 38, 53, 62, 76, 95, 110, 156, 279, and 388 (Fig. 1Go). Other functional motifs, such as the KLK motif at the N terminus of the third endoloop and the RLAK motif (35) immediately in front of the TMD 6, are also conserved in the receptor.



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  		Figure 1. The nucleotide sequences of gfGLP-1R cDNA, numbered from 5' to 3' relative to the ATG site (designated as +1), with predicted amino acids shown above the sequence in single-letter code. Seven predicted TMD regions are underlined and indicated. Three potential N-linked glycosylation sites and nine conserved cysteine residues are marked by the symbols # and *, respectively. A putative polyadenylation sequence (AATAAA) is shown in boldface, and the stop codon is also indicated. Arrows show the location and orientation of primers that were used in RT-PCR analysis for detecting the receptor expression.

 
Functional characterization of the gfGLP-1R
To determine the identity of the cloned receptor, several peptides belonging to the glucagon/secretin family were preliminarily used to stimulate the receptor in the cAMP assays. The results showed that, at 100 nM concentration, only hGLU, GLP-1 (7–36)amide human GLP-1 (hGLP-1), and human GIP (hGIP) were able to enhance the generation of intracellular cAMP levels in COS-7 cells transfected with the receptor, whereas other peptides such as secretin, vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) were devoid of stimulatory effects (data not shown). Subsequently, hGLU, hGLP-1, and hGIP were shown to be able to stimulate dose-dependent cAMP responses in the transfected cells, and their EC50 values were determined to be 1.2 nM, 0.9 nM, and 6.5 nM, respectively (Fig. 2IGo). To further demonstrate that the receptor is a functional gfGLP-1R, gfGLP-1 and gfGLU peptides were synthesized and used to stimulate the receptor. The results clearly showed that both peptides were potent agonists for this receptor (Fig. 2IIGo), and the EC50 values for gfGLP-1 and gfGLU were determined to be 0.18 nM and 0.53 nM, respectively. Hence, among all the peptides tested, gfGLP-1 was the most potent agonist identified for the receptor.



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  		Figure 2. cAMP assays of the gfGLP-1R, functionally expressed in COS-7n cells. Dose-dependent cAMP responses, using human peptides (I) and goldfish peptides (II), are shown. Data were obtained from three independent experiments and are expressed as mean ± SEM.

 
Tissue distribution of the gfGLP-1R
Northern blot analysis and RT-PCR were performed to study the tissue distribution of the gfGLP-1R. No hybridization signal was detected in the Northern blot (data not shown). This could possibly be attributable to the relatively low sensitivity of the method and/or low expression level of the receptor. Human GLP-1 receptor transcript was detected only in the pancreas by Northern blot analysis (6), whereas it was found in the pancreas, lung, brain, stomach, kidney, and heart by nuclease protection assay (16), which is a more sensitive method for detecting gene expression. Moreover, rat GLP-1 receptor transcript was detected in the lung, hypothalamus, hippocampus, cerebral cortex, kidney, pancreas, and throughout the gastrointestinal tract by RT-PCR analysis, but it could be found only in the lung, stomach, and large bowel by nuclease protection assay (17). In the present study, in addition to Northern blotting, a more sensitive technique, RT-PCR, was employed. Our results showed that the gfGLP-1R transcript was widely detected in various tissues, including the brain, gill, male gonad, lower and upper intestines, kidney, liver, heart, pituitary, muscle, and spleen (Fig. 3IGo). The data indicate that there are some tissues, such as the brain, intestine, heart, and kidney, where the GLP-1 receptor is expressed in both goldfish and mammals. Particularly, the receptor expression was found in 6 of 8 different regions in the goldfish brain, namely the olfactory bulbs and tracts, telencephalon, hypothalamus, medulla, spinal cord, and pituitary (Fig. 3IIGo). In a similar study, the expression of rat GLP-1 receptor was also found in numerous brain regions, such as the olfactory bulb, hypothalamus, and spinal cord (36). In the light of the findings of GLP-1’s action on the hypothalamus to inhibit food and water intake in rats (37) and catfish (38), it is possible that the GLP-1 receptor expressed in the hypothalamus of goldfish may also play a regulatory role in feeding.



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  		Figure 3. Tissue distribution of the gfGLP-1R. I, Detection of the receptor transcripts by RT-PCR in various tissues, including the brain (1), gall bladder (2), gill (3), male gonad (4), female gonad (5), lower intestine (6), upper intestine (7), kidney (8), liver (9), heart (10), pituitary (11), muscle (12), and spleen (13). A, Southern blot analysis of the PCR products using 32P-labeled gfGLP-1R cDNA probe. B, Amplification of the ß-actin cDNA using the same first-strand cDNAs as controls. II, Detection of the receptor expression by RT-PCR in different regions, including the olfactory bulbs and tracts (1), telencephalon (2), hypothalamus (3), optic tectum-thalamus (4), cerebellum (5), medulla (6), spinal cord (7), and pituitary (8). A solid arrow indicates the PCR products of the receptor cDNA with a size of 1135 bp, whereas a broken arrow indicates the products of the ß-actin cDNA with a size of 200 bp. Lanes C and M represent the negative control PCR and 1-kb DNA size marked (Life Technologies), respectively.

 
Identification of key residues in the extracellular domains of the gfGLP-1R
Functional characterization of the gfGLP-1R showed that several peptides with significant structural difference from gfGLP-1 were potent agonists for the receptor. These data suggest that the receptor is structurally flexible and can accommodate and interact with different peptide ligands, all of which are restricted to the structures found within the vertebrate glucagon/GLP-1/GIP/exendin sequences. To identify the residues within the putative ligand-binding domains of the gfGLP-1R that are responsible for peptide interaction, 11 mutant receptors, which contained substitutions in either the NT extracellular domain or the first exoloop, were constructed and functionally characterized. The protein alignment of the gfGLP-1R with other mammalian GLP-1, glucagon, and GLP-2 receptors (Fig. 4Go) served as a basis for the design of the mutants. They could be categorized into 2 types. The first type was constructed by substituting residues Q112D113D114 (also found in the zfGLP-1R) and R197 with the corresponding conserved residues in mammalian GLP-1 receptors, whereas the second type (D205 to A or K) was constructed by mutating the residue that is conserved in all the known GLP-1 receptors except zfGLP-1R. These amino acid residues are highlighted in Fig. 4Go.



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  		Figure 4. Protein alignment of the gfGLP-1R with GLP-1R in mouse, rat, human, zebrafish, glucagon receptor (GLU-R) in frog, mouse, rat, and human, and glucagon-like peptide-2 receptor (GLP-2R) in rat and human. The following regions, including the distal NT extracellular domain, TMDs 1–3, and the first exoloop are only shown and indicated in the figure. Residues that were selected for mutations in this study are highlighted in reverse mode. Numbers on the right show the positions of amino acids counted from the left, and the consensus sequence of these receptors is shown below the alignment. The protein alignment was generated by the GeneWorks program (Intelligenetics, Inc.).

 
Previous studies have shown that the extracellular domains of the GLP-1 (23, 24) and glucagon receptors (39, 40) are important for ligand binding. More importantly, charged amino acids present in these regions have been indicated to play essential roles. For these reasons, we selected the charged residues of the gfGLP-1R for mutations analysis within the NT extracellular domain. A triple mutant QDD(112–114)ESK was generated by substituting the QDD with the ESK motif, which is conserved in all the mammalian GLP-1 receptors (Fig. 4Go). To evaluate the effect of mutation on receptor functions, four different ligands, including gfGLU, gfGLP-1, hGLP-1, and exendin-4, were used. cAMP responses of the triple mutant were significantly reduced (44%, 51%, 43%, and 31% reduction, respectively, Fig. 5Go). Subsequently, point mutants of the Q112D113D114 motif showed that the Asp-to-Ser substitution at position 113 severely impaired the receptor function, whereas the other two mutants, with substitution at either 112 or 114, showed normal cAMP responses (Fig. 5Go). These results strongly indicated that the Asp residue present at position 113 of the gfGLP-1R is critical for receptor function. A Northern blot analysis was performed and showed that the mutant and wt receptors have similar expression levels (data not shown). This data demonstrated that the changes in the cAMP responses are not attributable to different expression levels of the receptors.



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  		Figure 5. cAMP responses of the mutants that contained specific mutations of the Q112D113D114 motif. The triple mutant QDD(112–114)ESK showed 44%, 51%, 43%, and 31% reductions upon gfGLU, gfGLP-1, hGLP-1, and exedin-4 stimulations, respectively (*, P < 0.01). The single mutant D(113)S displayed little cAMP response to all four peptides, whereas the mutants Q(112)E and D(114)K had similar cAMP responses to the wt receptor. Data are expressed as percentage of wt, mean ± SEM (n = 3).

 
At position 197 within the first exoloop, a positively charged Arg residue is present; whereas in the same position, an aromatic Trp residue is present and conserved in other mammalian GLP-1 receptors. A glutamine residue is located in the same position of the zfGLP-1R. (Fig. 4Go). To determine the functional role of this R197 residue, five mutants were constructed by substituting the Arg with two nonpolar residues (Ile and Ala), a negatively charged residue (Asp), a polar residue (Met), and the aromatic Trp residue (which is conserved in mammalian GLP-1 receptors). The cAMP assays showed that three of these mutations (from Arg to Ile/Ala/Asp) had detrimental effects on the receptor, whereas the other two substitutions (from Arg to Met/Trp) did not significantly alter the receptor’s responses (Fig. 6IGo). In addition to the R197, another charged amino acid, D205, is also present in the first exoloop, and this residue is conserved in gfGLP-1R and mammalian GLP-1 receptor (Fig. 4Go). Two mutants, D (B(205)A and D(205)K, were generated. Functional studies demonstrated that the D(205)A mutant is totally nonfunctional, whereas the D(205)K mutant has significantly reduced cAMP responses upon gfGLU, gfGLP-1, hGLP-1, and exendin-4 stimulations (66%, 63%, 41%, and 44% reduction, respectively, Fig. 6IIGo). By Northern blot analysis, all these mutants were also demonstrated to have similar expression levels in CHO cells (data not shown).



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  		Figure 6. cAMP responses of the mutants that contained substitutions in the first exoloop. I, The mutants R(197)A, R(197)A, and R(197)D showed little cAMP response upon stimulations by all four peptides, whereas for the mutants R(197)M and R(197)W, they had no significant changes in the cAMP response to various peptides. II, The mutant D(205)A displayed little cAMP response, and the mutant D(205)K also showed 66%, 63%, 41%, and 44% reductions upon gfGLU, gfGLP-1, hGLP-1, and exedin-4 stimulations, respectively (*, P < 0.01). Data are expressed as percentage of wt, mean ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously, we have characterized a proglucagon cDNA from goldfish C. auratus that encodes both glucagon and GLP-1 sequences (22). In the present study, we have identified and functionally characterized a GLP-1 receptor from the same species. Interestingly, several peptides that are structurally different from gfGLP-1 (Fig. 7Go), including hGLP-1, gfGLU, and hGLU, are also potent agonists for this receptor. Previous studies have demonstrated that both N and C termini of the GLP-1 molecule are important for its function (41, 42, 43). Specifically, residues at positions 1 (His), 4 (Gly), 6 (Phe), 7 (Thr), and 9 (Asp) of the N terminus are important for receptor interaction. These critical residues are conserved in the N termini of most of the peptides listed in Fig. 7Go, including gfGLP-1, hGLP-1, hGLU, and exendin-4. Substitutions at these positions, such as Y1 and I7 for hGIP and S7 for gfGLU, could affect their interactions with their receptors. The Y6 for gfGLP-1 and the F6 in other peptides probably have little effect, because both residues are structurally similar. In contrast, these peptides differ significantly in their C termini (Fig. 7Go). For instance, the last three residues of goldfish and hGLUs (MNS and MNT, respectively) are completely different from the last five residues of gfGLP-1 and hGLP-1 (KSGQP and VKGRG, respectively). Despite the structural differences of these peptides, they all act as potent agonists for the gfGLP-1R. Studies on mammalian GLP-1 receptor have shown that all other peptides of the glucagon superfamily bind poorly or are unable to bind to the receptor, with the exception of glucagon, which is a weak agonist with a binding affinity at least 100-fold less than that of GLP-1 (7, 44, 45). Taken together, these data suggest that the gfGLP-1R is structurally more accommodating than its mammalian counterparts in interacting with different peptide ligands, such as gfGLP-1 and hGLP-1 and glucagons. The fact that the gfGLP-1R is able to interact with both gfGLP-1 and glucagon indicates that these endogenous hormones may perform similar biological activities via the same receptor. This idea is consistent with previous findings that GLP-1 has a glucagon-like activity in fish to oppose the functions of insulin by stimulating gluconeogenesis and glycogenolysis (18).



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  		Figure 7. Peptide sequence comparison of gfGLP-1 with hGLP-1, gfGLU, hGLU, exedin-4, and hGIP. The peptides are numbered on the bottom of the alignment, and differences are highlighted in reverse mode.

 
Within the NT extracellular domain of the gfGLP-1R, a negatively charged Asp residue, D113 (Fig. 4Go), was identified to play an important functional role. Substituting it with a polar residue (Ser) that is conserved in other mammalian GLP-1 receptors completely abolished the cAMP responses to various peptides [mutant D(113)S, Fig. 5Go]. The loss of function of this mutant was likely caused by an impairment of ligand-receptor interaction rather than a failure of cell surface expression of the receptor. This argument is based on the fact that the triple mutant QDD(112–114)ESK retained 50% of the wt cAMP responses (Fig. 5Go). This triple mutant should have a complete loss of function if the same substitution at position 113 were already sufficient to disrupt cell surface expression. Moreover, data obtained from a previous study of the rat GLP-1 receptor suggested that mutations of residues present in the extracellular regions, but not in the TMDs, would not affect cell surface presentation of this receptor (24). In summary, our results clearly indicate that the negatively charged D113 residue, which is present in the NT extracellular domain of the gfGLP-1R, is a critical determinant for receptor function.

In the first exoloop, a positively charged Arg residue, R197 (Fig. 4Go), was also identified as a critical residue for receptor function. Mutation of this residue to Ile, AL, or Asp rendered the receptor to be completely defective. On the other hand, mutation of this residue to a polar Met or an aromatic Trp did not alter the receptor function (Fig. 6IGo). These data strongly suggest that the nature of the side chain of this amino acid residue is critical for ligand interaction. An amino acid with a bulky side chain, which is positively charged/polar/aromatic, seems to be essential at position 197 for ligand-receptor interaction. Our finding of this key residue in the gfGLP-1R is similar to that reported from a previous study of the rat GLP-1 receptor (24). At the position similar to that of this R197 residue, a negatively charged Asp residue is present and conserved in other mammalian GLP-1 receptors (Fig. 4Go), and this Asp residue has been shown to be important for high-affinity ligand binding (24). Taken together, our results indicate that the presence of the Arg or Met or Trp residue at position 197 of the gfGLP-1R is required for ligand-receptor interaction. Interestingly, the substitution of Arg to Trp (found in mammalian GLP-1 receptors) can be tolerated in the gfGLP-1R.

Another charged residue present in the first exoloop, D205 (Fig. 4Go), was also identified to be critical for receptor function. This Asp residue is conserved in gfGLP-1R and mammalian GLP-1 receptor (Fig. 4Go). Interestingly, in rat GLP-1 receptor, mutation of this residue to Ala had little effect on the receptor function (24). In gfGLP-1R, substitution of it with Ala or Lys dramatically hampered receptor functions (Fig. 6IIGo). Although this Asp residue is conserved in gfGLP-1R and rat/mammalian GLP-1 receptor, its functional role seems to be different among these receptors.

As mentioned earlier, specific amino acid residues of the GLP-1 molecule have been demonstrated to be important for receptor interaction (41, 42, 43). These critical residues include a positively charged His residue at position 1 (H1) and a negatively charged Asp residue at position 9 (D9). These residues are conserved in all the peptides listed in Fig. 7Go except hGIP, which contains a Tyr at position 1 (His->Tyr). From the present study, we have identified two negatively charged Asp residues (D113 and D205) and one positively charged Arg residue (R197) of the gfGLP-1R that are critical for receptor function. Mutation of these charged residues resulted in either a total loss or a dramatic reduction in receptor functions. Based on these observations, we speculate that these charged residues present in the extracellular domains of the gfGLP-1R are important for stabilizing the ligand-binding pocket of the receptor and/or involved in direct interaction with the charged amino acids H1 and D9 of the peptides used in this study, including gfGLU, gfGLP-1, hGLP-1, and exendin-4. All of these peptides have disordered structures in aqueous solutions and acquire more rigid conformations only upon the interaction with their membrane spanning receptors.

Our results indicate that the formation of the active center of the GLP-1 receptor is a multistep process. The initial and critical interaction requires a contact between the ligand and the GLP-1 receptor. This contact, perhaps a salt-bridge formation, is established between several individual amino acids from the ligand and several charged residues from the NT extracellular domain and the first exoloop of the GLP-1 receptor. This is followed by conformational changes in the ligand and receptor structures, leading to the formation of the binding pocket. In the case of the gfGLP-1R, the binding pocket can accommodate the structures of gfGLP-1 and hGLP-1, as well as their glucagon molecules. In contrast, the binding pocket of the mammalian GLP-1 receptor can accommodate only the structure of mammalian GLP-1 and not of glucagon. The ability of gfGLP-1R to bind to a wide range of peptide structures may primarily be a function of the initial interaction between the peptide ligands and the charged amino acids in the receptor, including D113 in the NT extracellular domain and R197 and D205 in the first exoloop.

In summary, we have isolated and functionally characterized a GLP-1 receptor from goldfish. Several peptides that have significant structural differences from gfGLP-1 were shown to be potent agonists of the receptor, suggesting that the gfGLP-1R is structurally more accommodating than its mammalian counterpart. Moreover, three charged residues, including D113 in the NT extracellular domain and R197 and D205 in the first exoloop, were identified to be critical for receptor function. These charged residues are structurally essential and may represent key residues responsible for interaction with the ligands.


   Footnotes
 
This work was supported by Grants HKU7181/99M, CRCG 335/026/0053, and 10203410 (to B.K.C.C.) from the Hong Kong Government Research Grants Council and by Grant IBN-9904506 (to S.M.) from the National Science Foundation.

Abbreviations: CHO, Chinese hamster ovary; gf, goldfish; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucagon-like peptide; GLP-1R, GLP-1 receptor; GLU, glucagon; GPCR, G-proteincoupled receptor; h, human; NT, N-terminal; PACAP, pituitary adenylate cyclase-activating polypeptide; TMD, transmembrane domain; VIP, vasoactive intestinal polypeptide; wt, wild-type; zf, zebrafish.

Received July 8, 2002.

Accepted for publication August 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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