Characterization of Glucagon-Like Peptide-1 Receptor ß-Arrestin 2 Interaction: A High-Affinity Receptor Phenotype

Rasmus Jorgensen, Lene Martini, Thue W. Schwartz and Christian E. Elling

7TM Pharma A/S (R.J., L.M., T.W.S., C.E.E.), Horsholm, DK-2970, Denmark; and Laboratory for Molecular Pharmacology (T.W.S.), The Panum Institute, University of Copenhagen, DK-2100, Denmark

Address all correspondence and requests for reprints to: Christian E. Elling, Ph.D., 7TM Pharma A/S, 2970 Horsholm, Denmark. E-mail: cee{at}7tm.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To dissect the interaction between ß-arrestin (ßarr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and ßarr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that ßarr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered ßarr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with ßarr2. However, when the wild-type receptor was coexpressed with both ßarr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with ßarr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of ßarr interaction between family A and B receptors also at the molecular level.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SEVEN TRANSMEMBRANE (7TM) G protein-coupled receptors interact with heterotrimeric G proteins upon activation. This results in activation of G protein-mediated signal transduction cascades leading to up- or down-regulation of intracellular levels of, for example, cAMP, inositol 1,4,5-triphosphate, or Ca2+. The termination of the receptor-mediated signaling is regulated by kinase activity phosphorylating the receptor, which leads to recruitment of intracellular scaffolding proteins such as ß-arrestins (ßarrs) from the cytosol, thereby excluding the receptor from further G protein interaction. Upon interaction with a 7TM receptor, ßarrs unmask domains that interact with a number of intracellular proteins (for review see Ref. 1) including the clathrin-coated pit components, adaptor protein-2 and clathrin, thereby recruiting the ßarr-interacting receptor to internalization (2).

According to the classical ternary complex model, the receptor has high affinity for agonists when in complex with a G protein. An increasing number of reports on rhodopsin-like family A receptors suggests that when ßarr interacts with an activated receptor, this likewise results in a stabilization of a distinct high-affinity conformation of the receptor (3, 4, 5, 6, 7). Fusion constructs in which the C-terminal tail of the receptor is fused to the N terminus of a G protein or ßarr have been used to explore the pharmacology of receptors in forced physical proximity to such specific interaction partners (3, 8, 9, 10) (for review on receptor-G protein fusions see Ref. 11). Tethering of the receptor to a downstream interaction partner ensures physical proximity between the two potentially interacting proteins, but does not necessarily exclude endogenously expressed proteins from interacting with the receptor (12). In this way the G protein/ßarr may interact with and stabilize transient conformations of the receptors that would not quantitatively recruit G proteins/ßarr unless stabilized by a ligand. Fusion constructs have therefore been used to characterize distinct high-affinity receptor conformations. In the tachykinin NK1 receptor, fusion to ßarr1 has been shown to create a monocomponent high-affinity receptor phenotype (3). Whereas these are well-established techniques within family A rhodopsin-like receptors, little or no results are available for family B glucagon-like receptors. Most members of the family B 7TM receptor subfamily are promising targets for pharmaceutical intervention. However, these receptors, so far, have proven intractable for classical small molecule drug discovery. Few pharmacological tool compounds therefore exist for these receptors. The glucagon-like peptide-1 (GLP-1) receptor is a family B 7TM receptor (for review see Ref. 13). As opposed to family A receptors, in which most endogenous agonists are expected to have their major contact points within the transmembrane helical domains, the endogenous ligands for family B receptors appear to have their major contact points in the large N-terminal domain, characteristic of family B 7TM receptors, with fewer intimate contacts to the transmembrane helical domain compared with family A receptors (14, 15, 16, 17). The predominant active form of the endogenous ligand for the GLP-1 receptor is GLP-1(7–36)amide (hereafter GLP-1), which increases glucose-dependent insulin secretion from pancreatic islets (18). Together with other actions, all leading to increased glycemic control, this makes the GLP-1 receptor a very promising target for treatment of type 2 diabetes using GLP-1 receptor agonists.

Here we demonstrate that the GLP-1 receptor interacts with ßarr2 and use fusion constructs to characterize the receptor phenotype pharmacologically. Interestingly, the fusion proteins have an attenuated G protein-mediated signal and display an increased apparent affinity selectively for one of the tested peptide ligands. We demonstrate that the pharmacological phenotype observed in the fusion construct can be obtained by coexpressing both ßarr2 and G protein-coupled receptor kinases (GRKs) with the wild-type receptor. Thus, like family A receptors, the GLP-1 family B receptor is stabilized in a high-affinity conformation by interaction with ßarr2.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GLP-1 Receptor-ßarr2 Fusion Protein Has Increased Affinity for Glucagon
The interaction of family A 7TM receptors with ßarrs is known to affect the pharmacological phenotype observed, including signaling and signal switching, internalization, and conformational freedom. To investigate the interaction between the GLP-1 receptor and ßarr2, we engineered fusion constructs in which the C terminus of the human GLP-1 receptor was fused directly to human ßarr2. Fusion constructs were made with either wild-type ßarr2 or ßarr2[R393E; R395E]. The [R393E; R395E] double mutation disrupts ßarr2 binding to adaptor protein-2 in the clathrin-coated pits (19, 20), resulting in an increased apparent surface expression of the GLP-1 receptor-ßarr2-[R393E; R395E] fusion construct compared with the GLP-1 receptor fused to wild-type ßarr2, as determined by Bmax measurements (Table 1GoGo). The GLP-1 receptor-ßarr2 fusion constructs were tested in parallel with the wild-type GLP-1 receptor in whole-cell competition binding using [125I]GLP-1, or [125I]exendin(9–39) as radioligands with unlabeled GLP-1, exendin-4, exendin(9–39), or glucagon. Exendin-4 is a 39-amino acid peptide found in the venom of the Gila monster Heloderma suspectum. It shares approximately 50% sequence identity with GLP-1 and is a full agonist on the GLP-1 receptor (21). Exendin(9–39) is a N-terminally truncated version of exendin-4 that lacks the first eight amino acids of exendin-4 and, as a consequence, functions as an antagonist on the GLP-1 receptor (21). Glucagon, the endogenous ligand for the related glucagon receptor, acts as a full agonist on the GLP-1 receptor although with low affinity and potency (22).


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Table 1. Whole-Cell Competition Binding with GLP-1 Receptor (GLP-1R) Fusion Constructs

 

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Table 1A.
 
Using [125I]GLP-1 as radioligand, the GLP-1 receptor-ßarr2 fusion constructs had unaltered affinities for GLP-1, exendin-4, and exendin(9–39) compared with the wild-type receptor (Fig. 1Go, A–C, and Table 1GoGo). Interestingly, the fusion constructs had up to 10fold increased apparent affinity for glucagon compared with the wild-type receptor [12 ± 1 nM for GLP-1 receptor-ßarr2[R393E; R395E]; 26 ± 3 nM for GLP-1 receptor-ßarr2 vs. 138 ± 23 nM for wild-type GLP-1 receptor (Fig. 1DGo and Table 1GoGo)]. An identical approximately 10-fold increase in apparent affinity for glucagon was observed using the antagonistic [125I]exendin(9–39) radioligand (Fig. 1HGo and Table 1GoGo) with no changes in affinity for GLP-1, exendin-4, or exendin(9–39). To ensure that the increased affinity for glucagon was not a nonspecific effect arising from the fusion of any substantial protein to the C terminus of the GLP-1 receptor, we tested whether a GLP-1 receptor-Renilla luciferase (GLP-1 receptor-Luc) fusion had an increased affinity for glucagon. The GLP-1 receptor-Luc fusion had a virtually unchanged affinity for GLP-1 and glucagon compared with the wild-type GLP-1 receptor [0.47 ± 0.17 nM vs. 0.49 ± 0.09 nM and 207 ± 137 nM vs. 138 ± 23 nM, respectively (Table 1GoGo)], indicating that the increased affinity for glucagon in the receptor ßarr2 fusions arose from a specific interaction between the receptor and ßarr2 as opposed to any protein of substantial molecular mass (Luc has a Mr of 36 kDa vs. 46 kDa for ßarr2). Thus, independent of whether a peptide agonist or antagonist radioligand was used, the GLP-1 receptor-ßarr2 fusion proteins had increased affinity for glucagon and unchanged affinity for GLP-1, exendin-4 and exendin(9–39) compared with the wild-type receptor.



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Fig. 1. Competition Binding on the Wild-Type GLP-1 Receptor (WT GLP-1R) and ßarr2 Fusion Constructs with Four Ligands and Two Radioligands

The unlabeled ligands used are GLP-1 (panels A and E), exendin(9–39) (panels B and F), exendin-4 (panels C and G), and glucagon (panels D and H). Two different radioligands were used: [125I]GLP-1 (panels A–D) and [125I]exendin(9–39) (panels E–H). Results are the means ± SEM of duplicate determinations in at least three independent experiments.

 
Coexpression of GLP-1 Receptor with ßarr2 and GRK5 Mimic the Pharmacological Phenotype of the Fusion Proteins
To test whether the increased apparent affinity for glucagon could be observed outside the context of the fusion proteins, we tested the wild-type GLP-1 receptor coexpressed with either ßarr2, GRKs, or both, because the activity of GRKs is a prerequisite for the formation of a receptor-ßarr complex. First, we measured the ability of the G{alpha}s-coupled GLP-1 receptor to mediate ligand-induced cAMP accumulation in transiently transfected COS-7 cells when the receptor was coexpressed with either GRK2, 3, or 5 to identify the GRK most likely to regulate the GLP-1 receptor. In all cases a decrease in efficacy and potency were observed compared with receptor expressed alone (Fig. 2Go). The most pronounced effect was obtained with GRK5, and this was chosen for further studies. Importantly, when the GLP-1 receptor was coexpressed with both ßarr2 and GRK5, a 10-fold increased apparent affinity for glucagon was observed [24 ± 6 nM vs. 247 ± 61 nM, respectively (Table 2Go and Fig. 3Go)], similar to the increase observed for the fusion proteins, suggesting that the molecular phenotype could be mimicked by coexpression of the separate proteins defining the fusion proteins combined with GRK5. This effect was observed only upon coexpression of the receptor with both ßarr2 and GRK5, and not by any two combinations of receptor with these separately (Table 2Go and Fig. 3Go). Similar to the observation in the fusion proteins, the affinity for GLP-1 was virtually unchanged in all cases.



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Fig. 2. GLP-1-Induced cAMP Accumulation

Dose-response curves with GLP-1 receptor (GLP-1R) cotransfected with vector, GRK2, GRK3, or GRK5. Results are the means ± SEM of duplicate determinations in three independent experiments.

 

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Table 2. Whole-Cell Competition Binding with GLP-1 Receptor (GLP-1R) Coexpressed with Combinations of ßarr2 and GRK5

 


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Fig. 3. Competition Binding on the Wild-Type GLP-1 Receptor (GLP-1R) Coexpressed with ßarr2 and/or GRK5

Dose-response curves with [125I]GLP-1 as the radioligand and GLP-1 (panel A) or glucagon (panel B) as the unlabeled ligand. Results are the means ± SEM of duplicate determinations in at least three independent experiments.

 
The C-Terminal Part of Glucagon Contains a Determinant for the High-Affinity Binding to the GLP-1 Receptor-ßarr2 Complex
To identify glucagon peptide determinants for the observed increased affinity toward the GLP-1 receptor-ßarr2 complex, we tested two chimeric GLP-1/glucagon peptides in whole-cell competition binding with the GLP-1 receptor-ßarr2-[R393E;R395E] fusion and wild-type GLP-1 receptor. The GLP-1 receptor-ßarr2-[R393E;R395E] fusion and the wild-type GLP-1 receptor both displayed the same affinity for the chimeric peptide A (3.8 ± 1.2 nM vs. 3.0 ± 0.35 nM, respectively) consisting of the first 14 amino acids from glucagon and the last 16 amino acids from GLP-1 (Fig. 4Go, A and B), indicating that the first 14 amino acids from glucagon do not contribute to the increased glucagon affinity for the GLP-1 receptor-ßarr2-[R393E;R395E] fusion. Interestingly, compared with the wild-type receptor, the GLP-1 receptor-ßarr2-[R393E;R395E] fusion had an increased affinity for the chimeric peptide B (0.5 ± 0.061 nM vs. 1.7 ± 0.34 nM, respectively) consisting of the first 26 amino acids from glucagon and the C-terminal four amino acids from GLP-1 (Fig. 4Go). Combined with the results for peptide A, this suggest that amino acids 15–26 of glucagon may contain a determinant for the increased affinity for the GLP-1 receptor-ßarr2-[R393E;R395E] fusion displayed selectively by glucagon. The relative increase in binding affinity does not parallel that of full-length glucagon, suggesting that a further contribution from the last three residues of glucagon or from a slightly different fold of the wild-type peptide may further add to the observed effect.



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Fig. 4. Competition Binding with Chimeric GLP-1/Glucagon Peptides

A, Primary structure of the chimeric peptides A and B and of the native peptides GLP-1 and glucagon (* describes a native peptide). Dose-response curves with peptide A (panel B) or peptide B (panel C) as the unlabeled ligand. Results are the means ± SEM of duplicate determinations in at least three independent experiments. GLP1R, GLP-1 receptor.

 
G Protein-Mediated Signal Transduction by the GLP-1 Receptor-ßarr2 Complex Is Attenuated
The GLP-1 receptor, like other family B receptors, signals strongly through a G{alpha}s-pathway leading to accumulation of cAMP. Whereas the Emax of the GLP-1 receptor-ßarr2-[R393E;R395E] fusion was significantly decreased for GLP-1 compared with the wild-type receptor (Emax of 11 ± 0.69 fmol cAMP/105 cells vs. 43 ± 3.2 fmol cAMP/105 cells, respectively) the control fusion construct GLP-1 receptor-Luc was only slightly reduced (Emax of 36 ± 5.3 fmol cAMP/105 cells vs. 43 ± 3.2 fmol cAMP/105 cells, respectively) despite a Bmax similar to the GLP-1 receptor-ßarr2-[R393E;R395E] fusion (Fig. 5AGo and Table 3Go). The potency of the GLP-1 receptor-ßarr2 fusions were 9- to 15-fold decreased compared with the wild-type receptor (0.37 ± 0.050 nM or 0.56 ± 0.13 nM vs. 0.040 ± 0.0057 nM, respectively) whereas the potency of the GLP-1 receptor-Luc control construct was only 4-fold decreased compared with the wild-type receptor (0.16 ± 0.020 nM vs. 0.040 ± 0.0057 nM, respectively) (Fig. 5AGo and Table 3Go). Hence, the GLP-1 receptor-ßarr2 fusion proteins had a significant decrease in both efficacy and potency compared with both the wild-type receptor and a control fusion construct with a similar surface expression as the receptor-ßarr2 fusion proteins. Because ßarr2, by interacting with the receptor, is expected to prevent further G protein coupling, this suggests that ßarr2 protein fused to the C terminus of the receptor specifically inhibits the functional coupling to the G protein. However, it is interesting to note that the fusion construct still retains some signaling properties (3, 12).



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Fig. 5. Ligand-Induced cAMP Accumulation

Dose-response curves for GLP-1-induced cAMP accumulation with wild type GLP-1 receptor (GLP-1R), GLP-1 receptor-Luc, GLP-1 receptor-ßarr2, or GLP-1 receptor-ßarr2[R393E;R395E] (panel A). B, GLP-1 receptor cotransfected with vector, ßarr2, GRK5, ßarr2 and GRK5, or GLP1R-ßarr2[R393E;R395E]+vector. Inset in panel B shows dose-response curves for glucagon-induced cAMP accumulation for GLP-1 receptor cotransfected with vector or ßarr2 and GRK5. Results are the means ± SEM of duplicate determinations in at least three independent experiments.

 

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Table 3. GLP-1 Induced cAMP Accumulation

 
To analyze further whether the observed inhibition of the functional coupling was governed by the physical proximity invoked from tethering the ßarr2 to the C terminus of the GLP-1 receptor, cAMP measurements were performed on the GLP-1 receptor coexpressed with either ßarr2, GRK5, or both (Fig. 5BGo). Coexpressing the wild-type receptor with ßarr2 did not influence the G{alpha}s receptor signaling. Importantly, coexpression with GRK5 resulted in an approximately 4-fold decrease in potency (EC50 of 0.24 ± 0.035 nM vs. 0.064 ± 0.0049 nM, respectively) and a 2-fold decrease in efficacy (Emax of 22 ± 3.8 fmol cAMP/105 cells vs. 51 ± 3.6 fmol cAMP/105 cells, respectively) (Fig. 5Go and Table 3Go). Coexpression with both ßarr2 and GRK5 (GLP-1 receptor + ßarr2 + GRK5) further resulted in a decrease in efficacy compared with coexpression with only GRK5 (Emax of 14 ± 2.1 fmol cAMP/105 cells vs. 22 ± 3.8 fmol cAMP/105 cells, respectively) making it identical to the response of the GLP-1 receptor-ßarr2-[R393E;R395E] fusion (EC50 of 0.26 ± 0.06 nM vs. 0.35 ± 0.01 nM, and Emax of 14 ± 2.1 fmol cAMP/105 cells vs. 16 ± 1.4 fmol cAMP/105 cells, respectively) (Fig. 5BGo and Table 3Go). A similar attenuation of glucagon signaling, through ßarr2 and GRK5 coexpression, was observed (Fig. 5BGo, inset). However, given the relatively modest potency of glucagon for the GLP-1 receptor, a quantitative assessment of this was difficult. Thus, whereas coexpression of ßarr2 with the wild-type GLP-1 receptor has no effect on GLP-1 receptor G protein-mediated cAMP signaling, a signaling phenotype similar to that of the GLP-1 receptor-ßarr2-[R393E;R395E] fusion can be obtained by coexpressing the wild-type GLP-1 receptor with both ßarr2 and GRK5, comparable to the results obtained in competitive binding.

Internalization of the Wild-Type and GLP-1 Receptor-ßarr2 Fusion Construct
GRK phosphorylation of activated receptors, leading to ßarr binding, will usually recruit the complex to ßarr-mediated internalization. Measurement of the agonist-induced internalization of the GLP-1 receptor-ßarr2 fusion construct demonstrated an increase in maximal receptors internalized compared with wild-type GLP-1 receptor (Fig. 6Go). In contrast, in agreement with both the binding and cAMP data, coexpression of the wild-type GLP-1 receptor with ßarr2 did not significantly increase the observed agonist-induced internalization. However, coexpression of GRK5 or ßarr2 and GRK5 significantly increased the agonist-induced maximal internalization compared with the wild-type GLP-1 receptor to a level comparable to the GLP-1 receptor-ßarr2 fusion construct (Fig. 6Go). As seen for the G protein-mediated signaling, the availability of GRK5, rather than ßarr2, appears to be the determining or limiting factor in obtaining a phenotype corresponding to a GLP-1 receptor-ßarr2 fusion construct. Again, the data suggest that the GLP-1 receptor-ßarr2 fusion construct mimics the phenotype invoked from coexpressing individual relevant signaling molecules.



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Fig. 6. Ligand-Induced GLP-1 Receptor (GLP-1R) Internalization

Percent internalized receptor after 30 min stimulation. Results are the means ± SEM of duplicate determinations in at least three independent experiments. *, P < 0.05 by t test.

 
Assessment of the Ability of the GLP-1 Receptor to Recruit ßarr2
The observation that tethering ßarr2 to the GLP-1 receptor attenuates receptor signaling and increases internalization, combined with the results showing that this phenotype may be mimicked by coexpressing ßarr2 and GRK5, suggests that a specific interaction between the receptor and ßarr2 is obtained in the ßarr2 fusion proteins and that this interaction is similar to that obtained from invoking their interaction though coexpression of the individual components of the signaling complex. To characterize this specific interaction, we tested the effect of GRK5 coexpression in a ßarr2 recruitment-based bioluminescence resonance energy transfer (BRET) assay. This is a proximity-based assay that measures energy transfer from a Luc-tagged protein to a green fluorescent protein (GFP) tagged protein upon addition of the Luc substrate, deep blue sea (23). We measure BRET2 using the GFP2 mutant variant of GFP. Upon substrate addition, Luc emits light at a wavelength of 400 nm. If GFP2 is within a distance of approximately up to 100 Å it is excited and emits light at a wavelength of 515 nm. The BRET2 signal is then measured as the ratio 400 nm:515 nm (23). The C terminus of the GLP-1 receptor was fused to Luc, and the N terminus of ßarr2 was fused to GFP2. The radioligand binding and signaling properties of the GLP-1 receptor-Luc fusion were assessed (Tables 1GoGo and 3Go) to ensure that the fusion of Luc did not change the pharmacological phenotype compared with the wild-type receptor. The two fusion constructs were coexpressed in COS-7 cells in a ratio that was expected to ensure overabundance of GFP2-ßarr2 relative to GLP-1 receptor-Luc. To ensure a robust BRET2 signal, we used a ßarr2-[R393E;R395E] double mutant, similar to the one used in one of the GLP-1 receptor-ßarr2 fusion constructs, in which the adaptor protein 2 interaction site has been mutated (20). GLP-1 induced ßarr2 recruitment with an EC50 of 2.61 ± 1.60 nM and an Emax of 43.7 ± 4.4 mBRET (Fig. 7Go). Interestingly, coexpression of GRK5 increased the Emax 2-fold to 102 ± 12 mBRET, with a preserved baseline signal, and with an unchanged EC50 of 3.28 ± 0.17 nM compared with receptor without GRK5 coexpression, indicating a potentiation of ßarr2 recruitment upon GRK5 coexpression. Glucagon, which had an increased affinity for the GLP-1 receptor-ßarr2-fusion protein and for the GLP-1 receptor coexpressed with ßarr2 and GRK5, was a low-potency full agonist on the GLP-1 receptor (Fig. 7Go). However, upon coexpression with GRK5, a significant increase in potency of glucagon was observed (82.6 ± 38.9 nM). Consistent with the previous observations, this indicates that GRK5 potentiates the recruitment of ßarr2 to the receptor, which in turn leads to stabilization of the receptor in a conformational state that has high affinity toward glucagon and unaltered affinity toward GLP-1.



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Fig. 7. ßarr2 Recruitment-Based BRET2 Assay with and without GRK5 Coexpression

GLP-1 or glucagon dose-response curves for ßarr2 recruitment as indicated in the legends. Results are the means ± SEM of duplicate determinations in at least three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In recent years there has been an increased attention on the interaction between 7TM receptors and ßarrs and the consequences of this interaction in terms of receptor conformations and signaling. The interaction with ßarrs has been shown to terminate not only receptor G protein coupling but also to initiate alternative signaling pathways such as MAPK pathways (for recent review see Ref. 24). At the same time, ßarrs appear to stabilize high-affinity conformations of 7TM receptors, which have led to the proposal of an alternative ternary complex model in which ßarrs can substitute for G proteins (5). Interestingly, it has been shown recently that ßarr interaction is also regulated by scaffolding proteins (25). The available data on receptor-ßarr interaction has been focused on family A receptors, and although family B receptors have been reported, in several cases, to recruit ßarrs, as evidenced by microscopy (26, 27, 28, 29, 30, 31, 32), this interaction has not been characterized in detail. In the present study, we show that in analogy to family A receptors, the family B GLP-1 receptor recruits ßarr2, resulting in a high-affinity molecular phenotype.

High-Affinity Glucagon Binding to GLP-1 Receptor-ßarr2
Previously, a fusion construct similar to the one described here, between the family A NK-1 receptor and ßarr1, has been used to demonstrate a high-affinity receptor phenotype of the receptor-ßarr complex (3). In the neurokinin-1 (NK-1) receptor fusion construct, the homologous binding curve for the endogenous agonist, substance P, had an unchanged affinity whereas other heterologous combinations of radioligands and unlabeled ligands had varying increases in affinity. Interestingly, a qualitatively similar result is obtained for the family B GLP-1 receptor-arrestin fusion construct. Whereas the homologous binding curve for the endogenous agonist GLP-1 had an unchanged affinity, glucagon had an apparent increase in affinity in competition with either radiolabeled GLP-1 or exendin(9–39). In the NK-1 receptor fusion construct, the most dramatic changes were observed when a radiolabeled nonpeptide antagonist, with its major mapped contact points in the exterior end of the transmembrane helices, was used in combination with peptide agonists (33). Unfortunately, no small molecule ligands are available for the GLP-1 receptor. Competition binding using two chimeric GLP-1/glucagon peptides suggested that the C-terminal, but not the N-terminal, half of the glucagon peptide contained a determinant for the increased binding affinity to the GLP-1 receptor-ßarr2 fusion. In contrast to family A receptors, family B receptors contain a large N-terminal domain that may be expressed independently of the transmembrane domain while retaining significant binding affinity (14, 15, 34). It has been proposed that the N-terminal eight amino acids of GLP-1 bind to the receptor transmembrane domain including connecting loops leading to receptor activation (14, 22). The C-terminal 22 amino acids of GLP-1 are proposed to bind to the large extracellular N-terminal domain of the GLP-1 receptor, and the most binding energy, by far, originates from this interaction (14). Exendin-4 gets an even greater proportion of the binding energy from this interaction through additional contacts to the receptor N-terminal domain (14, 15). The present study suggests that ßarr interaction induces or stabilizes a conformation of the receptor transmembrane domain, which is transmitted to or sensed by the N-terminal domain of the receptor, e.g. through contacts between the two domains, enabling glucagon to bind with increased affinity. Alternatively, or in addition, it has been suggested that the binding of the peptide C-terminal part to the receptor N-terminal domain may direct the nature of how the ligand peptide N terminus is presented to the receptor transmembrane domain (14). GLP-1 and exendin-4 interaction with the receptor N-terminal domain may orient the peptides in a way preventing them from exploring additional interaction sites in the transmembrane domain of the receptor-ßarr2 complex. As the antagonist exendin(9–39) is a truncated form of exendin-4 lacking the N-terminal eight residues, it does not significantly bind to the receptor transmembrane domain (14). Thus, detection of a ßarr-induced high-affinity family B receptor phenotype will therefore require the availability of ligands with binding characteristics allowing exploration of the receptor transmembrane domain. It would be expected that small molecule agonists binding to the transmembrane domain would have increased affinity for the receptor-ßarr complex. Further studies may shed more light on the mechanistic basis for the high-affinity phenotype.

Interestingly, the GLP-1 receptor-ßarr2 phenotype showing high-affinity binding for glucagon could also be obtained by coexpressing the wild-type GLP-1 receptor with both ßarr2 and GRK5 indicating that the fusion protein mimics the natural interaction of arrestin with the receptor. Studies on 7TM receptor-G protein fusions have suggested that tethering the G protein to the receptor serves mainly to bring the G protein into proximity of the membrane (and receptor) as opposed to ensuring a specific interaction with the cognate receptor (12). The current data suggest that the phenotype observed for the fusion proteins is originating from linking the arrestin to the membrane-proximal part but it does not discern whether a specific interaction with the cognate receptor is achieved per se or whether the linked arrestin may interact with the receptor of another fusion protein.

G Protein-Mediated Signal Transduction of the ßarr2 Fusion Proteins
It has been reported that agonist-induced desensitization of GLP-1 receptors, either in transfected CHL fibroblasts or endogenously expressed in insulinoma cells, leads to a decrease in both efficacy and potency of GLP-1-induced cAMP accumulation (35). Consistent with this, the signaling properties of the GLP-1 receptor-ßarr2 fusion proteins fusion, believed to mimic the GLP-1 receptor-ßarr2 complex, were characterized by a decrease in efficacy to approximately 25% of the wild-type GLP-1 receptor and approximately 30% of a control GLP-1 receptor-Luc fusion, the latter having a similar Bmax as the GLP-1 receptor-ßarr2 fusion proteins (Table 3Go and Fig. 4AGo). At the same time, the potency was reduced approximately 10-fold compared with the wild-type receptor. Interestingly, an approximately 10-fold reduction in EC50 for inositol phosphate turnover, the primary G protein-mediated signaling pathway for the NK-1 receptor, with an Emax of approximately 3% of wild type, was observed in the NK-1-arrestin fusion (3). The NK-1 receptor is known to have a very high affinity for ßarrs (36). Hence, the even more pronounced attenuation of the signaling observed in the NK-1 receptor compared with the GLP-1 receptor fusion proteins may reflect the different degree of transient dissociation of the receptor-ßarr complex between the GLP-1 receptor and the NK-1 receptor. Collectively, the signaling phenotype of the GLP-1 receptor-ßarr2 fusion proteins are characterized by a reduction in both efficacy and potency compared with the wild-type receptor, consistent with what was observed for the similar fusion of the family A NK-1 receptor and, as would be expected, for a desensitized GLP-1 receptor.

Interestingly, the phenotype observed with the GLP-1 receptor-arrestin fusion proteins could be obtained by coexpressing both ßarr2 and GRK5 with the wild-type GLP-1 receptor. As opposed to the binding experiments, in which the phenotype was observed only when both ßarr2 and GRK5 were coexpressed with the wild-type receptor, in the cAMP signal transduction experiments, GRK5 appeared to be the major determining or limiting factor. Overexpression of ßarrs has been shown to reduce signaling for a range of family A receptors, e.g. the ß2-adrenergic receptor (37) and family B receptors, e.g the secretin receptor (38), gastric inhibitory polypeptide receptor (39), PTH-1 receptor (40, 41), PTH-2 receptor (26), and vasoactive intestinal type-1 receptor (29). The stronger dependency on GRK5 could suggest that GRK5-mediated phosphorylation is sufficient to uncouple the GLP-1 receptor from G protein-mediated signaling, whereas the phenotype observed in binding further requires the recruitment of ßarr2 to the receptor, which in turn may be responsible for inducing or selecting the conformation having increased affinity for glucagon.

ßarr2 Recruitment and Internalization
Several factors, including both GRKs and second messenger kinases such as protein kinase A and protein kinase C (PKC), appear to regulate the internalization of family B receptors. Further, in a number of family B 7TM receptors, ßarrs can regulate receptor signaling without affecting internalization (26, 27, 30). In the gastric inhibitory polypeptide receptor, which is closely related to the GLP-1 receptor, it has been shown that receptor internalization in COS-7 cells is not affected by coexpressing the receptor with ßarr1 alone or in combination with any of GRK2, GRK5, or GRK6 (39), whereas agonist-induced cAMP accumulation is reduced by overexpression of GRK2. In the PTH-1 receptor, PKC activity is required for internalization without affecting ßarr2 recruitment, whereas GRK2 activity, although reducing agonist-induced signaling, does not influence internalization (41, 42). For the PTH-2 receptor, PKC has been suggested to control internalization (26). For the secretin receptor it has been shown that overexpression of ßarrs and/or GRKs does not enhance receptor internalization but that protein kinase A activity controls internalization (30). Nonetheless, overexpression of GRKs does inhibit secretin receptor signaling. Other family B receptors, such as the calcitonin receptor-like receptor, has been shown to have ßarr-mediated internalization (28). Hence, the picture of the factors involved in the internalization of family B receptors is thus ambiguous, and it seems that second messenger-regulated kinases can just as well regulate receptor internalization as can GRK phosphorylation/ßarr interaction. Clearly, an observed effect of GRK overexpression on receptor signaling does not ensure a parallel effect on receptor internalization.

The GLP-1 receptor-ßarr2 fusion showed a marked increase in internalization compared with the wild-type receptor as would be expected if internalization of the receptor can be mediated by the fused ßarr2. Coexpressing the wild-type GLP-1 receptor with ßarr2 did not cause any significant increase in receptor internalization whereas coexpression with GRK5 increased receptor internalization to the level of the GLP-1 receptor-ßarr2 fusion. Coexpressing the GLP-1 receptor with both ßarr2 and GRK5 did not increase the internalization further. Hence, as observed in the cAMP experiment, the determining or limiting factor for the internalization phenotype is again GRK5, presumably leading to recruitment of endogenously expressed ßarrs and subsequent ßarr-mediated internalization. To directly measure the GLP-1 receptor/ßarr2 interaction and the influence of GRK5 on this interaction, we used a ßarr2 recruitment-based BRET2 assay. We found that GLP-1 induces recruitment of GFP2-ßarr2 to the GLP-1 receptor-Luc fusion construct in a dose-dependent manner. GRK5 coexpression potentiated the maximal GLP-1-induced BRET2 signal by 133%. Glucagon was a full agonist with a low potency compared with GLP-1. Interestingly, GRK5 coexpression enhanced the EC50 for glucagon-induced GFP2-ßarr2 recruitment, as would be expected from the binding experiments, although direct quantification was made difficult by the low potency without GRK5 coexpression. However, the fold difference in affinity/potency between GLP-1 and glucagon was reduced to a similar magnitude in the ßarr2 recruitment assay upon GRK5 coexpression, as observed in the binding assay on the GLP-1 receptor-ßarr2 fusion, i.e. an approximately 30-fold difference. Whether this has any physiological relevance is unknown, but it is interesting to note that ßarr2 is known to initiate MAPK signaling upon interaction with 7TM receptors and that GLP-1 receptors are thought to stimulate a number of cellular responses through activation of MAPK pathways (43).

According to the classical model for GRK involvement in 7TM receptor/ßarr2 interaction, GRK-mediated phosphorylation of the receptor tail will break the polar core of ßarrs, thereby increasing the affinity for the activated 7TM receptor (44). Whereas GRK5 coexpression is required for obtaining the full phenotype when receptor, ßarr2, and GRK5 are expressed separately, the GLP-1 receptor-ßarr2 fusion constructs appear to circumvent the requirement for exogenous GRK5 and, through the tethering, achieve the ßarr2-bound conformation. Further experiments will reveal, in more detail, the underlying mechanisms.

In conclusion, we propose that the GLP-1 receptor-ßarr2 fusion mimics the natural GLP-1 receptor-ßarr2 complex described by an increased affinity for glucagon but not other peptide ligands tested, and with a phenotype that further is characterized by attenuated G protein-mediated signaling and increased internalization. We hypothesize that such constructs may be useful as tools in discovering, for example, small molecule agonists for this class of receptors that have so far proven intractable for classical small molecule drug discovery.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular Biology
Human ßarr2 N-terminally tagged with GFP2 (GFP2-ßarr2) and the Renilla luciferase (Luc) cDNA was purchased from PerkinElmer, Wellesley, MA. Mutations [R393E;R395E] in the ßarr2 part were introduced in human GFP2-ßarr2 using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Human GLP-1 receptor cDNA was cloned from a pancreatic cDNA library (CLONTECH, Palo Alto, CA) and inserted into pcDNA3.1+ (Invitrogen, San Diego, CA). GLP-1 receptor cDNA lacking the stop codon was made by standard molecular biology techniques and subcloned in a 5'-position of ßarr2 and ßarr2-[R393E;R395E], respectively. The C-terminally Luc-tagged GLP-1 receptor construct was made using standard molecular biology techniques. All cDNA clones were verified by sequencing. Bovine GRK2, GRK3, and GRK5 cDNA were inserted in pcDNA1.

Transfections and Tissue Culture
Cloned receptors were transiently expressed in COS-7 cells transfected by calcium phosphate precipitation according to previously reported methods (45). For cotransfections of receptor DNA together with ßarr2 and/or GRK5, a 1:4:1.5 ratio of cDNA was used. This same 1:4:1.5 ratio was used for GLP-1 receptor-Luc + GFP2-ßarr2[R393E;R395E] + GRK5 transfections for the BRET assay. Vector without insert was used to ensure a constant amount of total DNA in all compared transfections. COS-7 cells were obtained from the European Collection of Animal Cell Cultures. The cells were routinely maintained and passaged in complete DMEM supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 2 mM Glutamax-I, and 10 µg/ml gentamicin (all from Invitrogen Life Technologies, Carlsbad, CA) and incubated at 37 C in a 90% humidified atmosphere of 10% (vol/vol) CO2 in air.

Competition Binding
Transfected cells were transferred to 24-well poly-D-lysine-treated culture plates at a density aiming at 5–10% binding. Two days after transfection, the cells were assayed by competition binding for 3 h at 4 C using approximately 15 pM [125I]GLP-1(7–36) amide (Amersham Biosciences, Buckinghamshire, UK) or [125I]exendin(9–39) (PerkinElmer Corp., Norwalk, CT) plus variable amounts of unlabeled ligand in 25 mM Tris-HCl, 5 mM MgCl2 buffer (pH 7.4) supplemented with 0.1% (wt/vol) BSA, and 40 µg/ml bacitracin. The nonspecific binding was determined as the binding in the presence of the highest peptide concentration indicated. All experiments were performed in duplicate determinations. GLP-1, glucagon, exendin-4, and exendin(9–39) were obtained from Bachem (Bubendorf, Switzerland). The chimeric peptides were made as previously described (46). Competition binding data were analyzed and IC50 values determined by nonlinear regression analysis using Prism (GraphPad Software, Inc., San Diego, CA). Values of the dissociation (Kd) and inhibition (Ki) constants and number of binding sites (Bmax) were estimated from competition binding using the equations Kd = IC50 – L and Bmax = B0 x IC50/L, where L is the concentration of radioligand and B0 is specific bound radioligand.

Determination of Intracellular cAMP Accumulation
Agonist-induced cAMP accumulation was measured as previously described (45). Briefly, COS-7 cells were seeded in 24-well culture dishes 1 d after transfection at a density of 100,000 cells per well and supplemented with 2 µCi [3H]adenine/ml (Amersham Pharmacia Biotech, Piscataway, NJ). Two days after transfection, cells were washed twice with HBS (25 mM HEPES; 0.75 mM NaH2PO4; 140 mM NaCl, pH 7.2) and incubated in HBS buffer supplemented with 1 mM 3-isobutyl-1-methylxanthine. Variable concentrations of peptide were added, and the cells were incubated for 45 min at 37 C. The assay was terminated by aspirating the buffer followed by addition of ice-cold 5% trichloroacetic acid. cAMP was isolated by applying the supernatant to a 50W-X4 resin (Bio-Rad Laboratories, Inc., Richmond, CA) followed by an alumina resin (A-9003, Sigma Chemical Co., St. Louis, MO). Determinations were made in duplicate.

ßarr2 Recruitment Assay
BRET2 measurement was done as previously described (20) using a Mithras LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany). Briefly, after harvesting, 180 µl of resuspended COS-7 cells were distributed in 96-well microplates (white Optiplate; PerkinElmer, Wellesley, MA) resulting in a density of approximately 200,000 cells per well. Agonist was added manually, and substrate addition was performed with an injector that injected the substrate DeepBlueC (final concentration, 5 µM; Packard Bioscience) 2 sec before reading. The optimal reading time after agonist addition was determined to be 5 min. The signals detected at 400 nm and 515 nm were measured sequentially, and the 515:400 ratios calculated.

Receptor Internalization Using Radioactive Agonist
One day after transfection COS7-cells were split in 24-well poly-D-lysine-treated culture plates at a density of 200,000 cells per well. Agonist-induced endocytosis was quantified 2 d after transfection using [125I]GLP-1(7–36)amide. First, cells were washed once in prewarmed media (DMEM containing 0.1% BSA) and then incubated in media containing approximately 70 pM radioligand at 37 C for 30 min. To stop the reaction the cells were quickly washed twice with ice-cold PBS and incubated in ice-cold 50 mM acetic acid containing 150 mM NaCl (pH 2.8) on ice for 12 min to separate acid-labile (interpreted as cell surface) from acid-resistant (interpreted as internalized) radiolabeled ligand. Internalized radioactivity was measured in cell lysates after incubation in 200 mM NaOH solubilization buffer containing 1% sodium dodecyl sulfate. Nonspecific binding was measured in the presence of 100 nM unlabeled ligand and was subtracted to give specific binding. Internalization was defined as the fraction of internalized radioactivity (present in the lysate from solubilized cells) to total radioactivity (acid removable and acid resistant) for each data point. Experiments were performed in duplicate determinations.


    ACKNOWLEDGMENTS
 
We thank Robert J. Lefkowitz (Duke University Medical Center, Durham, NC) for kindly providing the GRK cDNAs.


    FOOTNOTES
 
First Published Online November 4, 2004

Abbreviations: ßarr, ß-Arrestin; BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; GLP, glucagon-like peptide; GRK, G protein-coupled receptor kinase; Luc, Renilla luciferase; NK-1, neurokinin-1; PKC, protein kinase C; 7TM, seven transmembrane.

Received for publication August 4, 2004. Accepted for publication October 28, 2004.


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