Scanning of the Glucagon-Like Peptide-1 Receptor Localizes G Protein-Activating Determinants Primarily to the N Terminus of the Third Intracellular Loop

Swarna K. Mathi, Yvonne Chan, Xinfang Li and Michael B. Wheeler

Departments of Medicine and Physiology University of Toronto Toronto, Ontario, Canada M5S 1A8


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It is well known that glucagon-like peptide-1 (7–36 amide) (tGLP-1) is a potent insulinotropic hormone with powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the intracellular domains of the tGLP-1 receptor that are required for its efficient coupling to adenylyl cyclase because cAMP is the primary candidate second messenger coupling tGLP-1 to insulin secretion. Recently, we identified an amino acid within the third intracellular loop, K334, that was required for efficient coupling of tGLP-1 receptor to adenylyl cyclase. A similar mutagenesis-based strategy was employed here to examine the first and second intracellular loops and to further define sequences in the third loop required for the efficient coupling of the receptor to its second messengers. Receptor mutants were expressed in COS-7 cells and examined for tGLP-1 binding and cAMP stimulation. Three alanine substitution mutations, V327A, I328A, and V331A, resulted in significantly lower tGLP-1-stimulated cAMP production without reductions in receptor expression. Analysis of the first and second intracellular loops revealed only one mutation contained within the first loop, R176A, where a significant reduction in cAMP activation was observed with normal receptor expression. These studies suggest that specific determinants of coupling for tGLP-1 receptor are primarily localized to the predicted junction of the fifth transmembrane helix and the third intracellular loop. We predict that V327, I328, and V331 form part of a hydrophobic face that directly contacts the G protein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The incretin hormone, glucagon-like peptide-1 (GLP-1), is released from the intestinal L cells to stimulate insulin secretion from pancreatic ß-cells in a glucose-sensitive manner. The biologically active forms secreted, truncated (t) GLP-1(7–37) and tGLP-1(7–36 amide), have been shown to be among the most potent insulinotropic agents identified to date in mammals (1). The recent observation, that mice expressing a null mutation in the GLP-1 receptor (R) are diabetic, clearly establishes a major role for GLP-1 in mammalian carbohydrate metabolism (2). A possible role for tGLP-1 in the central control of feeding has also recently been suggested (3). Truncated GLP-1 administered via an intracerebroventricular injection was a powerful inhibitor of feeding in fasted rats, a response proposed to be mediated through tGLP-1 receptors in the hypothalamus. In addition, clinical studies indicate that tGLP-1 not only stimulates insulin secretion in normal subjects, but also in those with non-insulin-dependent diabetes mellitus (4, 5, 6), supporting its therapeutic potential.

The insulinotropic properties of tGLP-1 are mediated through a high-affinity tGLP-1 receptor on the insulin-secreting ß-cells of the pancreas (7, 8). The receptor cDNA, initially cloned from rat pancreatic islet cells, and subsequently from human pancreas (9, 10), predicts a seven-transmembrane G protein-coupled receptor (GPCR) of the glucagon/vasoactive intestinal peptide (VIP)/secretin, receptor subfamily (11). Studies in monkey kidney COS cells have shown that the recombinant tGLP-1R can couple to at least two G protein-coupled signaling pathways, including adenylyl cyclase (AC) and phospholipase C (12). Other work on the endogenous receptor in isolated ß-cells and ß-cell lines, and with the recombinant receptor expressed in CHO (Chinese hamster ovary) and CHL (Chinese hamster liver) cells, however, strongly suggests that the cAMP-dependent activation of protein kinase A and subsequent increase in free cytosolic Ca2+ is the trigger for tGLP-1-stimulated insulin secretion (13, 14, 15).

Numerous investigations of ligand-activated signal transduction have centered on the localization of sites of contact between the receptor and proteins activated by the receptor. Structural studies on the GPCR superfamily suggest that the predicted ends of the transmembrane helices and the connecting intracellular loops present logical contact points with G proteins. In keeping with this hypothesis, each of the intracellular loops and the C-terminal domain have been implicated in receptor activation (16). Within the biogenic amine receptor family there is widespread consensus that the third intracellular domain (IC3) contains a G protein activation region (17, 18, 19). There is, however, significantly less information on potential G protein coupling within the glucagon/GLP-1 receptor subfamily. At present, receptor mutagenesis studies suggest that the C-terminal domain may be important for receptor regulation/desensitization, but a direct role of this domain in AC coupling is not supported (20, 21). The IC3 loop, however, has recently been implicated in G protein activation by the tGLP-1 receptor. A single amino acid block in the predicted N-terminal portion of the IC3 domain (K334-L335-K336) was shown to be required for the efficient coupling of tGLP-1R to AC (22). Within this block, the K334 position was found to be most important for coupling. Because direct interaction with G proteins most likely involves large protein interfaces (16), we examined the predicted first and second loops of the rat GLP-1 receptor and have further examined the junction between the predicted fifth transmembrane (TM5) helix and the IC3 loop. Our results suggest that residues within the cytoplasmic loops, the majority of which are concentrated at the junction between the TM5 helix and the IC3 domain, are required for transducing the tGLP-1 signal to cAMP activation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of the First Intracellular Loop
To examine the potential interaction between G proteins and the tGLP-1 receptor, a series of deletion and substitution mutants spanning the intracellular loops of the rat tGLP-1 receptor were generated, expressed in COS-7 cells, and analyzed for [125I]tGLP-1 binding and cAMP formation in response to a tGLP-1 stimulation. In all cases, basal cAMP levels (in the absence of tGLP-1 stimulation) were found to be similar, suggesting that no mutation created a constitutively active receptor. At a concentration of 10 nM (determined previously to yield a maximal cAMP stimulation), tGLP-1 elicited an approximately 30-fold increase in cAMP accumulation with the wild type (WT) tGLP-1 receptor, consistent with stimulation levels reported previously (12). Strikingly lower cAMP responses, compared with WT tGLP-1R, were observed with three deletion mutations, each carrying a three-amino acid deletion (IC1–1-3) spanning the predicted nine-amino acid IC1 domain (data not shown). For each mutation examined in this loop and subsequent regions, binding displacement curves were generated according to a one-site model, which described the data most accurately in all cases. Key descriptors of the binding analysis, the dissociation constant (Kd) and Bmax values, were calculated as described in Materials and Methods. For the WT tGLP-1R, the Kd value was in the range of 2.1–4.1 nM (see Figs. 1–4GoGoGoGo), which corresponds well with previous studies (12, 22). The deletion mutations spanning IC1, however, failed to demonstrate measurable specific binding, which likely explains the lack of a cAMP response (data not shown). To reduce the possibility of impairing the appropriate processing and/or expression of viable receptors, an alanine replacement strategy was employed to allow analysis of specific amino acid moieties with minimal alteration to receptor conformation. Further analysis using single amino acid substitutions showed varying levels of cAMP accumulation. COS cells expressing the alanine substitution mutants F169A, R170A, H171A, L172A, H173A, and T175A were capable of eliciting cAMP responses that were not found to be significantly different (P > 0.05) from the WT response (Fig. 1Go). In contrast, cAMP responses for C174A, R176A, and N177A were significantly different at 37 ± 5%, P < 0.05; 26 ± 8%, P < 0.01; and 43 ± 6%, P < 0.05, respectively, compared with WT levels. Competitive binding experiments were employed to determine whether appropriate cell surface expression could possibly explain the reduced cAMP accumulation. With mutants C174A and N177A, Bmax values were found to correlate well with reduced receptor activation because significantly lower values compared with WT (45 ± 6 and 22 ± 2% of WT, P < 0.01) were observed. In contrast, with L172A, H173A, and T175A, receptor expression (Bmax) was reduced, with little effect on cAMP accumulation, suggesting that the levels of receptor expression are not always linearly related to responsiveness. With mutation R176A, which produced the lowest cAMP response of the substitution mutants examined in IC1, the receptor number (111 ± 10% of WT) and the Kd value (5.5 ± 1.2 vs. 3.9 ± 0.3 nM for the WT) were not considered significantly different. Further, dose-dependent analysis of the cAMP response to tGLP-1 (10-12 through 10-6 M) with the R176A mutant (Fig. 5bGo, data for 10-6 M tGLP-1 not shown) indicated that the EC50 was approximately 10-fold higher (R176A = 22 nM vs. WT GLP-1R = 1.7 nM), suggesting that a specific modification at this basic residue in position 176 of IC1 impairs the stimulation of cAMP production.



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Figure 1. Comparison of WT tGLP-1R and Substitution Mutants of IC1 for Stimulation of cAMP Production and Receptor Expression

cAMP formation 72 h posttransfection in COS-7 cells stimulated by 10 nM tGLP-1 (as indicated by the vertical stippled bar) is shown as a percentage of WT. The amino acid substituted for alanine in each mutant is outlined within the sequence. The level of expression (Bmax) and binding affinity (kd) for each deletion mutant was calculated from binding displacement curves and expressed relative to the WT GLP-1R (% WT). Results are presented as the mean ± SEM of at least three independent experiments. Receptor mutations which led to significant alterations (P < 0.05) in cAMP stimulationa or expressionb are indicated. Mutations resulting in decreased cAMP production that are poorly correlated to changes in receptor expression are indicated by arrows.

 


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Figure 2. Comparison of WT tGLP-1R with Block Deletion and Substitution Mutants of the IC2 Region

Binding characteristics and cAMP activation of block deletion mutants (A) and alanine substitution mutants (B) of the IC2 region were determined in transiently transfected COS-7 cells as described in Fig. 1Go. Results are presented as the mean ± SEM of at least three independent experiments. Receptor mutations which led to significant alterations (P < 0.05) in cAMP stimulationa or expressionb are indicated. Detectable binding displacement was not observed with IC2–3 and 4 as indicated.

 


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Figure 3. Comparison of WT tGLP-1R with Mutants Containing Block Deletions at the Junction of the Fifth Transmembrane Region and the Third Intracellular Loop

cAMP accumulation in COS-7 cells transfected with block deletion mutants, DM1-DM6, as well as the level of expression (% Bmax) and binding affinity (% Kd), is expressed relative to the WT. Results are presented as the mean ± SEM of at least three independent experiments, and significant differences are indicated as in Figs. 1Go and 2Go. Arrows indicate mutations as described previously in Fig. 1Go.

 


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Figure 4. Comparison of WT tGLP-1R with Alanine Substitutions Mutants at the Junction of the TM5 Domain and the IC3 Loop

cAMP formation in COS-7 cells and binding characteristics are expressed as a percentage of WT. Values are shown as the mean ± SEM from three or more independent experiments, and significant differences are indicated as in Fig. 1Go. Arrows indicate mutations as described previously.

 


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Figure 5. Competitive Binding and cAMP Dose-Response Analyses of Receptors with Impaired cAMP Responses

A, The displacement of [125I]tGLP-1 binding in COS-7 cells, transiently transfected with WT and receptor mutants R176A, V327A, I328A, V331A, and DM-1 is expressed as specific binding. Results are representative of at least three independent experiments. B, Dose-dependent effects of tGLP-1 on cAMP accumulation in COS-7 cells. One representative of two independent experiments is shown.

 
Analysis of the Second Intracellular Loop
The second intracellular domain has also been implicated in G protein activation within the biogenic amine receptor family (23), further suggesting the possibility of multiple receptor/G protein contact points. To test the possibility that the residues within the tGLP-1 receptor IC2 domain contribute to the efficient coupling of the receptor to cAMP, the block deletion strategy was initially employed. Mutational analysis of the IC2 domain revealed that deletions in the distal portion of the predicted loop, IC2–3 and IC2–4, were poorly tolerated (Fig. 2aGo). Block deletion mutants IC2–1 and IC2–2 had reduced cAMP responses, the former reduction being statistically significant (32 ± 3% vs. WT, P < 0.01). Binding analysis, however, revealed a strong correlation between ligand-binding efficiency and cAMP production, suggesting that coupling was not specifically altered by these mutations (Fig. 2aGo). IC2–3 and IC2–4 were chosen for further examination using single amino acid substitutions because deletions were not tolerated in these blocks. Surprisingly, no substitution mutations resulted in a reduction in the cAMP response (Fig. 2bGo). There appeared to be, in fact, a trend toward an exaggerated cAMP response with two mutations, I265A and F266A. In each case the affinity and expression level of the receptor mutants were not considered significantly altered, relative to the unmodified receptor (Fig. 2bGo).

Analysis of the N-Terminal Junction of the Third Intracellular Loop
Previously, we reported that a single amino acid block deletion in the predicted N-terminal portion of the rat tGLP-1 receptor IC3 domain (K334-L335-K336) was required for the efficient coupling of tGLP-1R to AC (22). Since the N-terminal region may contain several residues that contribute to coupling, as predicted from studies by other members of the GPCR superfamily (17, 18), we further examined the junction between the predicted TM5 helix and the third cytoplasmic loop. Deletion mutants spanning A333-F321 (DM1-DM6) were also assayed for [125I]tGLP-1 binding and cAMP formation. Significantly lower cAMP responses compared with WT tGLP-1R were observed with all DM block deletion mutants (15–4% vs. WT, P < 0.01) with the exception of DM4 (Fig. 3Go). The binding affinities of mutants DM1-DM4 were not substantially altered, as determined by Kd values, although mutants DM1-DM3 did have significantly reduced receptor expression, with Bmax at approximately 38–25% of WT (P < 0.01). Importantly, the reduced expression levels with DM-1 and DM-3 do not correlate with the virtual lack of cAMP activation by these mutants, strongly suggesting an uncoupling from AC and implicating the involvement of residues within these deletions. The Kd and Bmax values could not be determined for DM5 and DM6 because appreciable displacement of the radioligand was not observed.

Further analysis by single amino acid substitution within this deleted region revealed three residues that severely impaired cAMP activation when modified, although not to the extent of the corresponding block deletion mutants (Fig. 4Go). The V327A, I328A, and V331A substitutions resulted in a significantly lower cAMP response compared with WT GLP-1 receptor (42 ± 6, 39 ± 5, and 45 ± 3%, respectively, P < 0.01) (Fig. 4Go). The reduced stimulation was not correlated to significant alteration in binding affinity or a reduction in the level of receptor expression relative to the WT (Figs. 4Go and 5aGo), suggesting the importance of these residues in coupling to AC. Further dose-dependent analysis of the V327A, I328A, and V331A mutants for cAMP production were performed to examine GLP-1-induced cAMP sensitivity (Fig. 5bGo). For each mutation examined, it was apparent that the EC50 value for cAMP stimulation was approximately 10-fold greater than the WT GLP-1R (V327 = 26 nM, I328 = 16 nM, V331 = 23 nM vs. WT = 1.7 nM), despite the observation that the affinity of each receptor for tGLP-1 binding was not significantly different (Fig. 5aGo). These data further suggest a less efficient coupling of the receptor mutants to G protein activation.

Sufficient evidence exists to suggest that the proximal IC3 domain of GPCRs may form an amphipathic {alpha}-helix (18, 24, 25, 26). To better understand the relationship between the block deletion mutants and the single substitution mutants, a helical representation of the N-terminal IC3 loop was generated, following the model of Hill-Eubanks et al. (18) for the muscarinic m5 receptor (Fig. 6Go). The critical amino acids, V327, I328, and V331, as well as K334 identified from our previous study, are localized to one face of the predicted {alpha}-helix (Fig. 6aGo). Because DM4 maintained normal phenotype with respect to ligand binding and AC activation, this deletion mutant is also represented in a helical projection that can be aligned with the WT tGLP-1R (Fig. 6bGo). The sequence of all deletion mutants is also given for comparison (Fig. 6bGo). DM2, -3, -5, and -6 mutants may be expected to have normal activation because they have compatible amino acid substitutions at the required positions. These mutants, however, have significantly reduced receptor expression, which may lead, at least in part, to significantly lowered activation.



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Figure 6. A Helical Projection of the IC3 N-Terminal Domain Comparing WT and Mutant Receptors

A, A view of the tGLP-1 receptor IC3 loop from the membrane to the interior of the cell is shown according to the model of Hill-Eubanks et al. (18). The open line represents the top of the view at the membrane surface. The sequence of the complete IC3 domain and the predicted junctions of TM5 and TM6 are also given in linear form. The critical residues indicated by circles on the helical view are shown in boxes on the linear view of IC3. The circled amino acid on the linear view represents the only single amino acid substitution we generated with completely abolished cAMP activation. B, A comparison of the WT sequence with the block deletion mutants D1-D6 is given to determine the effect of these deletions on the three-dimensional structure of the IC3 N terminus. Positions corresponding to the four critical residues of the WT receptor are also shown in a helical view of the DM4 mutant. These positions are also underlined within the sequence comparison of all deletion mutants. The symbol ++ indicates that DM4 elicits a normal cAMP response while all other deletions have blunted cAMP responses.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mechanism by which the binding of an agonist to a GPCR leads to activation of intracellular signaling systems is the focus of intense investigation. Evidence has accumulated indicating that the predicted intracellular domains of the GPCRs contain specific regions that provide contact points with G proteins. The C terminus (IC4) and the third intracellular cytoplasmic loop, which display the highest amount of heterogeneity among the GPCR family, have been implicated in G protein activation (16). Previously, we characterized the IC3 loop of the tGLP-1 receptor between residues K334-K351, using a series of deletion and substitution mutations scanning the region (22). A single amino acid, K334, in the N-terminal region of IC3, was required for the efficient coupling of the tGLP-1 receptor to AC, supporting the general model that the IC3 domain of GPCRs contains specific residues required to activate G proteins. Since K334 is located near the junction with the predicted TM5 region, and several amino acids are likely involved in direct protein-protein contact with G proteins (16), we have continued our analysis of this area into the predicted transmembrane domain. Analysis at this junction, spanning F321-A333, identified five deletion mutations that significantly lowered the cAMP response and receptor expression (Fig. 3Go). The reduced stimulation of cAMP accumulation with the deletion mutations DM-1 and DM-3, however, was far more dramatic than the corresponding reduction in receptor expression, suggesting that this region may contain key residues involved in G protein contact. Further analysis by single alanine substitution revealed three critical residues that, when modified, resulted in receptors with impaired signaling properties. V327A, I328A, and V331A substitutions resulted in a significant reduction in cAMP response without appreciable alteration in binding affinity or reduced expression relative to the WT receptor (Figs. 4Go and 5Go). Interestingly, these amino acids, along with K334 identified previously (22), are concentrated at the junction between TM5 and IC3.

Although it will not be possible to unequivocally define the precise junction between IC3 and TM5 without structural data, the existence of amphipathic {alpha}-helices at the N and C termini that are continuous with the predicted transmembrane helices is supported (24, 25, 26). Evidence that the N-terminal region of IC3 in the m5 muscarinic receptor exists as an {alpha}-helix with functional importance was provided using random saturation mutagenesis (18). This region within the m5 receptor contained four critical amino acids that are predicted to face one side of the amphipathic helix, terminating in a completely conserved, charged residue (Arg). If the IC3 region of tGLP-1 receptor is initiated at I325, the four residues identified in our studies are analogous, in position and type, with those of the m5 receptor (18). When this model is applied to the tGLP-1 receptor, it can be predicted to form a continuous {alpha}-helix that terminates at the charged lysine residue, suggesting that V327, I328, V331, and K334 form an analogous hydrophobic face, which couples directly with the G protein (Fig. 6aGo). In the m5 receptor, the outer residues of the hydrophobic face were proposed to be involved in direct ionic interaction with the G protein, whereas the inner residues were involved in maintaining the hydrophobic interactions that define the structure of the coupling pocket (27). Analysis of the amino acid sequence alignment for the entire family of glucagon/VIP/secretin receptors revealed only identical amino acids or conservative substitutions for the critical hydrophobic residues, terminating in a completely invariant lysine residue. Therefore, this subfamily of GPCRs predicts a three-dimensional structure for the N-terminal IC3 region consistent with the model proposed for the muscarinic receptors. The C-terminal region of IC3, as well as the N-terminal region, has also been implicated in direct G protein coupling with the muscarinic receptors (18, 26). We have not identified similar C-terminal residues within the tGLP-1R. Two separate mutations in this region at position 350, however (A350E or A350K), were not tolerated and thus could not be tested (22). Interestingly, Heller et al. (28) described a tGLP-1 receptor substitution, R348G, rather than the R348A mutant we described earlier (22), which resulted in a receptor with significantly reduced affinity for tGLP-1 and an abolished cAMP response. In our experience, mutations that dramatically compromise receptor expression or binding affinity are difficult to interpret with respect to alterations in G protein coupling. Thus, R348-L349-A350 and other residues within the intracellular domains may indeed play important roles in coupling and require further study.

The possibility that further G protein coupling determinants within the glucagon/tGLP-1 receptor family involve loops in addition to IC3 has been the focus of several recent studies. Deletion of the majority of the IC4 domain of the glucagon receptor did not alter the ability of glucagon to activate AC (20). Similar work with the tGLP-1R (21), and with the structurally related GIP (29) and PTH (30) receptors, demonstrate that large blocks of the predicted IC4 domain can be eliminated without uncoupling the receptor from AC. Although, these studies do not support the presence of direct G protein coupling within this domain, it appears that this region in each receptor may be important for receptor regulation through homologous and heterologous desensitization mediated through phosphorylation and receptor internalization (21). The involvement of the IC1 and IC2 domains in G protein activation within the glucagon receptor family has received little attention. We have generated a series of deletion and substitution mutations that encompasses the IC1 and IC2 domains (Figs. 1Go and 2Go). These studies revealed only one potential site within IC1, R176, where a notable reduction in tGLP-1-mediated cAMP stimulation was observed without a corresponding effect on receptor expression (Fig. 1Go). It is unclear how R176 may play a role in coupling along with residues in the proximal IC3 region; however, one can speculate that intracellular domains may be in close proximity and therefore could interact with the G protein. Another recently described mutation in the GLP-1R (H180R) (28), caused a dramatic shift in affinity and a parallel decrease in cAMP activation. We predict, however, that H180 is probably positioned within TM-2 and is not likely to provide a direct contact point for G proteins. The IC2 region appeared to lack any determinants of coupling and activation because no amino acid substitutions resulted in significantly impaired cAMP accumulation. This domain, in fact, had uncharacteristically exaggerated cAMP responses with some substitution mutations in the distal part of the loop.

In conclusion, we have extended our characterization of the junction between the fifth transmembrane domain and the third intracellular loop of the rat tGLP-1 receptor using a mutagenesis-based strategy. We have identified several mutations that produced receptors with reduced responsiveness to tGLP-1. Although it was not possible to fully ascertain whether the reduced activity of all mutants examined ( Fig. 1–4GoGoGoGo) was the result of reduced receptor expression and/or increased EC50 values, three amino acids localized to IC3 (V327, I328, and V331) displayed significantly decreased cAMP activation when mutated, without significant alterations to receptor expression. These residues, along with K334 (identified in our earlier work), therefore, may be required for binding G proteins and activating AC. Our findings are consistent with recent studies of the m5 muscarinic receptor, which provide strong evidence that the IC3 N-terminal region exists as an {alpha}-helix continuous with the TM5 domain, terminating with a conserved basic residue (18, 27). The four amino acids of the GLP-1 receptor that were critical for G protein coupling and activation of AC were found in analogous positions to those of the m5 receptor and could form a hydrophobic face when viewed in a helical arrangement of the region. The presence of highly conservative substitutions in corresponding positions of all other members of the glucagon/VIP/secretin subfamily, as well as receptors for various biogenic amines (27), suggests that a common mechanism of activation may be widely shared by diverse seven- transmembrane receptors coupled to G proteins. Based on the prediction that these residues are required for direct side chain interactions with the G protein, as well as the structural integrity of the hydrophobic pocket, we intend to test amino acid substitutions that specifically alter these properties in the future. In addition, since the levels of receptor expression are not always linearly related to responsiveness (35), several mutations with reduced Bmax and AC activation identified in the present study should receive further examination as well.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Receptor Mutants and Cell Transfection
The WT rat tGLP-1 receptor DNA (9) was introduced into the HindIII-XbaI sites of the vector pBS (Stratagene, La Jolla, CA) before mutagenesis. Mutations were introduced into the GLP-1 receptor coding sequence by oligonucleotide-directed mutagenesis using double-stranded template (Stratagene), or single-stranded template (31, 32). Mutated sites were verified by dideoxy sequencing with T7 polymerase (Pharmacia, Uppsala, Sweden). Mutant receptor cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA) for transient expression in COS-7 cells. Approximately 3 x 106 cells were seeded in 10-cm dishes and cultured in DMEM supplemented with 10% FBS. Cells were transfected with WT tGLP-1R or receptor mutants (5 or 10 µg for cAMP and binding assay, respectively), using the diethylaminoethyl-dextran method as previously described (12).

Iodination of GLP-1 and Binding Assays
Synthetic human tGLP-1(7–36) amide (Bachem, Torrance, CA) was used in all binding studies. Radioiodination was accomplished by the chloramine T method as previously described (12, 33). The [125I]tGLP-1(7–36 amide) product was purified by reverse phase adsorption to a C-18 Sep-pak column (Waters Associates, Milford, MA) and had a specific activity of approximately 125–250 µCi/µg. Whole cell binding assays were performed as previously described (12, 33). Briefly, COS-7 cells expressing the WT and mutant receptors were cultured for 72 h posttransfection, washed twice in PBS, and recovered from plates with 2 mM EDTA in PBS. Cells (~5 x 105/tube) were incubated for 45 min at 37 C in binding buffer (DMEM containing 0.4% glucose, 1% BSA, and 0.1 mg/ml bacitracin, pH 7.4) with radiolabeled tracer [125I]tGLP-1 amide (50,000 cpm, ~270 pM) and unlabeled peptide at concentrations of 10-14 to 10-6 M, in a final volume of 200 µl. Cell suspensions were centrifuged at 12,000 x g and the cell-associated radioactivity was counted (Cobra II, Canberra Packard, Meriden, CT). Specific binding (total binding minus nonspecific binding measured in the presence of excess (1 µM) tGLP-1) was determined for the WT and each mutant receptor. Binding characteristics, including specific binding, Bmax, and Kd, were calculated according to Keen and MacDermot (34) from competitive binding-displacement curves generated using Prism (GraphPad Software, San Diego, CA).

cAMP RIAs
COS-7 cells expressing the WT and mutant receptors were passaged into six-well plates 24 h posttransfection. After culture for an additional 48 h, cells were washed in PBS, followed by assay buffer (DMEM containing 0.4% glucose, 1% BSA, and 0.1 mg/ml bacitracin, pH 7.4), and preincubated 30 min at 37 C, followed by a 30-min stimulation period with tGLP-1 (10 nM) and 3-isobutyl-1-methylxanthine (1 mM). For dose-response curves in the 24-well format, cells were stimulated with tGLP-1 (10-12 to 10-6 M). The cAMP was extracted with 80% ethanol, and lyophilized samples were reconstituted in sodium acetate buffer (pH 6.2). Cyclic AMP production was measured by RIA (Biomedical Technologies, Stoughton, MA) as previously described (12, 33). All test agents were prepared from lyophilized samples on the day of assay and added from concentrated stocks.

Statistics
All values are expressed as the mean ± SEM of at least three independent observations unless stated otherwise. Statistical analysis was performed using ANOVA followed by Tukey’s post analysis (InStat, GraphPad Software, San Diego, CA), comparing each test condition to those values obtained with the WT tGLP-1R.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Patricia Brubaker for her expert advice with several technical aspects of the study and for supplying [125I]tGLP-1.


    FOOTNOTES
 
Address requests for reprints to: Michael B. Wheeler, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.

This work was funded by grants to M.B.W. from the Medical Research Council of Canada (MT-12898) and the Canadian Diabetes Association in memory of the late Marjorie and Willis E. Montgomery

Received for publication December 16, 1996. Revision received January 23, 1997. Accepted for publication January 23, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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