From the
Molecular Pharmacology, Novo Nordisk,
DK-2760 Maaloev, Denmark, the
Department of
Medicinal Chemistry, Pharmaceutical University of Denmark, DK-2100 Copenhagen,
Denmark, and ¶Medicinal Chemistry IV and
||Pharmacological Research 3, Novo Nordisk, DK-2760
Maaloev, Denmark
Received for publication, January 31, 2003 , and in revised form, April 15, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GluR and GLP-1R belong to family B of the 7TM GPCRs, which includes the receptors for peptide hormones of the glucagon/PACAP superfamily: glucagon-like peptide-2 (GLP-2), glucose-dependent insulinotropic polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), growth hormone-releasing hormone, and secretin and other peptide hormones such as calcitonin, corticotrophin-releasing factor, and parathyroid hormone (PTH). The fingerprint of family B 7TM GPCRs is six conserved cysteines that form three disulfide bonds in the N-terminal extracellular domain (Nt-domain) (3). The structural constraints imposed by an identical disulfide bond pattern probably guide the Nt-domain of family B 7TM GPCRs into a common structural fold regardless of limited sequence identity (4, 5). The present knowledge about the structure and arrangement of the 7TM helices is based primarily on multiple sequence alignment analyses, although a functional coupling of conserved polar residues of the PTH receptor (PTH1R) suggests the existence of a cooperative helix-helix interface between TM2 and TM7 (6, 7).
Structure-activity analyses of peptides of the glucagon/PACAP superfamily
suggest that their entire length is required for optimal biological activity
and that the C terminus is primarily involved in receptor binding, whereas the
N terminus contains the residues involved in receptor activation
(8). The N-terminally modified
glucagon analog desHis1Glu9-glucagon is a potent GluR
antagonist, which emphasizes the importance of His1 and
Asp9 of glucagon in activation of GluR
(9). The N termini of GLP-1 and
exendin-4 are highly conserved, and the N terminus of exendin-4 is essential
for activation of GLP-1R (10).
Furthermore, His7, Gly10, Phe12,
Thr13, and Asp15 of the GLP-1 N terminus are important
for optimal binding and activation of GLP-1R, and they are all conserved in
exendin-4 (11). NMR structures
of GLP-1, glucagon, and PACAP-(138) in lipophilic solvents or
dodecylphosphocholine micelles agree that the central and C-terminal parts are
-helical, often with a central distortion of the helix geometry,
whereas the N terminus is a flexible random coil
(1214).
Interestingly, the N terminus of receptor-bound PACAP-(121) forms a
specific
-coil structure, and the PACAP N terminus is important for
receptor activation (8,
14). The N termini of glucagon
and PACAP are highly conserved, and thus the glucagon N terminus may form a
PACAP-like structure upon binding to GluR.
The isolated Nt-domain of family B 7TM GPCRs is sufficient for low affinity ligand binding, and it is a critical determinant of ligand selectivity (3, 4, 15). The Nt-domain of GLP-1R binds exendin-4-(939) with high affinity, and therefore either the C-terminal extension of exendin-4 (the Trp cage) or divergent residues in exendin-4 and GLP-1 increase the affinity of exendin-4 at the Nt-domain of the GLP-1R relative to GLP-1 (16). Additional interactions with the extracellular loops (ECL) and the extracellular end of the 7TM helices may explain the high affinity ligand binding of intact receptors and provide additional determinants of ligand selectivity (1720).
The molecular information about receptor-ligand complexes of family B 7TM GPCRs is limited. A two-site binding model has been proposed for the ligand interaction with PTH1R, in which the PTH C terminus interacts with the PTH1R Nt-domain and the PTH N terminus interacts with the PTH1R core domain (21). Peptides of the glucagon/PACAP superfamily may follow a similar binding mechanism (14, 22). The conserved Asp198 in the boundary between TM2 and ECL1 of GLP-1R is important to maintain the binding site for the GLP-1 N terminus (23). Specifically, Asp3 in the N terminus of VIP and secretin interacts with positively charged residues in the extracellular end of TM2 of VPAC1 (VIP receptor) and the secretin receptor, respectively (24, 25). The corresponding Gln3 in the N terminus of glucagon most likely interacts with the extracellular end of TM2 of GluR (26). In addition, p-benzoyl-L-phenylalanine in position 22 or 26 of the secretin C terminus cross-links to specific residues in the Nt-domain of the secretin receptor (27).
Family B 7TM GPCRs selectively bind their natural ligands with high affinity, although they may bind homologous ligands with low affinity. GLP-1R binds GLP-1 with high affinity and glucagon with low affinity, and glucagon is a low potency full agonist of GLP-1R (22). The GLP-1R Nt-domain defines almost completely the glucagon/GLP-1 selectivity profile of GLP-1R by selective recognition of the GLP-1 C terminus, and the GLP-1R core domain is not important for glucagon/GLP-1 selectivity (22). In contrast, GluR has a very strong glucagon/GLP-1 selectivity profile and does not cross-react with GLP-1. The GluR Nt-domain selectively recognizes the glucagon C terminus, and the GluR core domain strongly determines specificity for the glucagon N terminus. These results encouraged a search for the selectivity determinants in the GluR core domain. Single-substituted glucagon analogs addressing four divergent residues in the glucagon and GLP-1 N termini were analyzed for their ability to bind and activate GluR. Loss-of-function mutations in glucagon were rescued by gain-of-function mutations in GluR. The results show that three distinct epitopes of the GluR core domain determine specificity for the N terminus of glucagon.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peptide Synthesis and RadiolabelingGlucagon, GLP-1-(737), Ala2-glucagon, Glu3-glucagon, Val10-glucagon, Ser12-glucagon, Ser8-GLP-1, Gln9-GLP-1, Tyr16-GLP-1, and Lys18-GLP-1 were synthesized and characterized as previously described (22).
The radioligand 125I-Glucagon (2.2 Ci/µmol) was prepared by the chloramine-T method, and 125I-GLP-1 (2.2 Ci/µmol) was prepared by the lactoperoxidase method (29). Both radioligands were purified by reverse-phase high pressure liquid chromatography.
Cell Culture and Transient Receptor ExpressionHEK293 cells were maintained in Dulbecco's modified Eagle' medium supplemented with 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (90 units/ml and 90 µg/ml, respectively). Cells were seeded in T75 flasks, transfected with 9 µg of DNA using the FuGeneTM transfection reagent (Roche Molecular Biochemicals), harvested 24 h after transfection, and used for plasma membrane preparations or applied directly to functional experiments.
Functional AssayHEK293 cells transiently expressing the desired wild type or chimeric receptor were harvested and resuspended in assay buffer (Flashplate®; PerkinElmer Life Sciences) to a cell density of 1.8 x 106 cells/ml. Peptides were diluted in phosphate-buffered saline with 0.02% Tween 20. Cells (50 µl) and peptides (50 µl) were mixed in 96-well Flashplates® (PerkinElmer Life Sciences), gently agitated for 5 min, and incubated for 25 min at room temperature. The resulting intracellular level of cAMP was measured according to the supplier's manual, and data were analyzed by nonlinear regression analysis using Prism®, (GraphPad Software, Inc.).
Binding AssayBinding experiments were performed as
previously described (22).
Briefly, a freshly thawed plasma membrane preparation of HEK293 cells
transiently expressing the desired wild type or chimeric receptor was
incubated for 2 h at 30 °C in the presence of peptide and tracer. Bound
and unbound peptide/radioligand were separated, and filters were washed twice
in cold incubation buffer and left to dry. Finally, the amount of bound
radioligand was determined using a Packard -counter, and data were
analyzed by nonlinear regression analysis using Prism®, (GraphPad
Software, Inc.).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Chimera AThe chimeric receptor, chimera A, consists of the GluR Nt-domain and the GLP-1R core domain (Fig. 1) (22). The single-substituted glucagon analogs were analyzed for their ability to bind and activate chimera A. In competition binding, glucagon displaced 125I-glucagon from chimera A with an IC50 value of 1.2 nM (Fig. 1C and Table I). The IC50 values of Ala2-glucagon and Glu3-glucagon were lower than that of glucagon, whereas the IC50 values of Val10-glucagon and Ser12-glucagon were similar to that of glucagon (Fig. 1C and Table I). In functional experiments with chimera A, all of the glucagon analogs were equipotent with glucagon (Fig. 1D and Table I). The results obtained with GluR and chimera A showed that substitution of the GluR core domain with the GLP-1R core domain increased the affinity and potency of Ala2-glucagon, Glu3-glucagon, Val10-glucagon, and Ser12-glucagon relative to glucagon.
Dissection of the Glucagon Receptor Core DomainSmall segments of the GluR core domain were substituted with the corresponding segments of GLP-1R. Ala2-glucagon, Glu3-glucagon, and Ser12-glucagon were analyzed for their ability to bind and activate three chimeric receptors: ChTM2, ChECL2, and ChECL3 (Fig. 1). In ChTM2, the extracellular end of TM2 of GluR was substituted with the corresponding segment of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChTM2 with an IC50 value of 1.4 nM (Fig. 1E and Table II). The IC50 value of Glu3-glucagon was lower than that of glucagon, whereas the IC50 values of Ala2-glucagon and Ser12-glucagon were higher than that of glucagon. On whole cells transiently expressing ChTM2, Glu3-glucagon was equipotent with glucagon, whereas Ala2-glucagon and Ser12-glucagon were less potent than glucagon (Fig. 1F and Table II). The results obtained with GluR and ChTM2 showed that substitution of the extracellular end of TM2 of GluR with the corresponding segment of GLP-1R increased the affinity and potency of Glu3-glucagon relative to glucagon, Ala2-glucagon, and Ser12-glucagon. In addition, the affinity but not the potency of Ala2-glucagon had increased relative to glucagon and Ser12-glucagon.
|
In ChECL2, the second extracellular loop and the extracellular ends of TM4 and TM5 were substituted with the corresponding segments of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChECL2 with an IC50 value of 3.1 nM (Fig. 1G and Table II). The IC50 value of Ser12-glucagon was similar to that of glucagon, whereas the IC50 values of Ala2-glucagon and Glu3-glucagon were higher than that of glucagon. On whole cells transiently expressing ChECL2, Ser12-glucagon was equipotent with glucagon, whereas Ala2-glucagon and Glu3-glucagon were less potent than glucagon (Fig. 1H and Table II). The results showed that substitution of the second extracellular loop and the extracellular ends of TM4 and TM5 with the corresponding segments of GLP-1R specifically increased the affinity and potency of Ser12-glucagon relative to glucagon, Ala2-glucagon, and Glu3-glucagon.
In ChECL3, the third extracellular loop and the extracellular ends of TM6 and TM7 were substituted with the corresponding segments of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChECL3 with an IC50 value of 1.9 nM (Fig. 1I and Table II). The IC50 value of Ala2-glucagon was lower than that of glucagon, whereas the IC50 values of Glu3-glucagon and Ser12-glucagon were higher than that of glucagon. On whole cells transiently expressing ChECL3, Ala2-glucagon was equipotent with glucagon, whereas Glu3-glucagon and Ser12-glucagon were less potent than glucagon (Fig. 1J and Table II). The results showed that substitution of the third extracellular loop and the extracellular ends of TM6 and TM7 with the corresponding segments of GLP-1R specifically increased the affinity of Ala2-glucagon relative to glucagon, Glu3-glucagon, and Ser12-glucagon. In functional experiments, the potency of Ala2-glucagon and to a lesser extent Ser12-glucagon had increased relative to glucagon and Glu3-glucagon.
We were unable to generate a chimeric receptor that specifically increased the affinity and potency of Val10-glucagon; therefore, Val10-glucagon was not analyzed further.
Point Mutations in the Glucagon ReceptorThe region defined
by the GLP-1R segment of ChECL3 was investigated by site-directed mutagenesis
of the corresponding segment of GluR. The divergent residues in this region
were substituted individually to the corresponding residues of GLP-1R.
Initially, the GluR point mutants were analyzed in binding and functional
experiments with glucagon and Ala2-glucagon. In competition binding
with the Asp385-Glu mutant, the affinity of
Ala2-glucagon was slightly higher than that of glucagon
(Fig. 2C and
Table II). In functional
experiments with whole cells transiently expressing the Asp385-Glu
mutant, the potency of Ala2-glucagon was slightly higher than that
of glucagon (Fig. 2D
and Table II). With the other
GluR point mutants, the affinity and potency of Ala2-glucagon were
lower than the affinity and potency of glucagon, in a manner similar to GluR
(Table II). This is illustrated
by the Ser379 Phe mutant
(Fig. 2, A and
B). Subsequently, the Asp385
Glu and
Ser379
Phe mutants were analyzed in binding and functional
experiments with Glu3-glucagon and Ser12-glucagon. At
both mutants, the affinity and potency of Glu3-glucagon and
Ser12-glucagon were significantly lower than the affinity and
potency of glucagon. The results showed that the point mutation
Asp385
Glu in the extracellular end of TM7 specifically
increased the affinity and potency of Ala2-glucagon relative to
glucagon, Glu3-glucagon, and Ser12-glucagon.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dissection of the GluR core domain identified three distinct epitopes of the GLP-1R core domain that rescued the affinity and potency of specific glucagon analogs. The extracellular end of TM2 (ChTM2) rescued Glu3-glucagon, ECL2 and the proximal segments of TM4 and TM5 (ChECL2) rescued Ser12-glucagon, and ECL3 and the proximal segments of TM6 and TM7 (ChECL3) rescued Ala2-glucagon. ChTM2, ChECL2, and ChECL3 did not compromise glucagon binding or potency, which confirmed the structural integrity of the chimeric receptors. Two nonexclusive explanations may account for these results: 1) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 interact directly with Glu3, Ser12, and Ala2, respectively, and/or 2) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 are required to maintain a local binding site conformation that accommodates the interaction with Glu3, Ser12, and Ala2, respectively. Nevertheless, it is difficult to explain the results without considering the proximity of the GLP-1R segments in ChTM2, ChECL2, and ChECL3 with Glu3, Ser12, and Ala2 of the glucagon analogs, respectively. In addition, the corresponding segments of GluR define the strong glucagon/GLP-1 selectivity profile of the GluR core domain by selective recognition of the glucagon N terminus.
Most family B 7TM GPCRs have two positively charged residues in TM2, whereas GluR has only one and a neutral hydrophobic residue in place of the other (Lys187 and Ile194 in human GluR). Furthermore, the glucagon/PACAP superfamily peptides have either Asp or Glu in position 3 except glucagon, which has a Gln. The K187R/I194K GluR mutant rescued both affinity and potency of Asp3-glucagon relative to glucagon (26). The additional positive charge of the K187R/I194K GluR mutant and the chimeric receptor ChTM2 probably accommodates the extra negative charge of Asp3-glucagon and Glu3-glucagon, respectively. In fact, Glu3-glucagon bound ChTM2 with higher affinity than glucagon but was equipotent with glucagon in functional experiments with ChTM2. The small discrepancy in affinity versus potency suggests that Glu3 of Glu3-glucagon provides a binding determinant for interaction with ChTM2 that is not equally favorable for activation of ChTM2. However, the discrepancy is small compared with the total rescue of Glu3-glucagon by ChTM2. The corresponding Asp3 of secretin and VIP interact with the conserved Arg and Lys in TM2 of the secretin and VPAC1 receptor, respectively, and the interaction with Arg in the center of TM2 is important for receptor activation (24, 25). Site-directed mutagenesis of a highly conserved His in the cytoplasmic end of TM2 leads to constitutive activity of several family B 7TM GPCRs (3032). Collectively, it appears that TM2 is important for agonist binding and activation of family B 7TM GPCRs. Ile194 in TM2 of GluR may serve as a selectivity determinant that prevents access of homologous peptides to the activation site of GluR.
The combined analyses of GluR mutants and single-substituted glucagon analogs demonstrated the functional significance of the correlated substitution of residues in glucagon (Gln3) and GluR (Ile194). Glucagon is highly conserved during evolution, and specifically position 3 is occupied by Gln in vertebrates except bony fish, where position 3 is occupied by either Gln, Asp, or Glu. In bony fish, glucagon and GLP-1 have overlapping biological activities, and GLP-1 acquired the incretin function after the divergence of bony fish and mammals (33). Specificity of receptor-ligand pairs most likely evolved to ensure distinct physiological functions, and therefore the change of selection pressure on position 3 of glucagon may reflect the divergence of the biological activities of glucagon and GLP-1.
Eight divergent residues in the region defined by the GLP-1R segment of
ChECL3 was investigated by site-directed mutagenesis of GluR. Only the
substitution of Asp385 with the corresponding Glu of GLP-1R rescued
the affinity and potency of Ala2-glucagon without disturbing the
pharmacological profile of the other glucagon analogs relative to glucagon. It
is difficult to explain the rescue of affinity of Ala2-glucagon by
a direct interaction with Glu385 in the Asp385
Glu GluR mutants. It seems more likely that the Asp385
Glu
mutation stabilized a local binding site conformation that preferably
interacted with Ala2-glucagon. Nevertheless the results strongly
suggest proximity between Ala2 of Ala2-glucagon and the
extracellular end of TM7 of the GluR mutant Asp385
Glu.
The dissection of the GluR core domain provides three constraints that orient the glucagon N terminus with respect to the structural elements of the GluR core domain. Given the structural integrity of the GluR mutants in this study, we suggest a binding model in which Ser2 of glucagon is close to Asp385 of TM7, Gln3 of glucagon is close to Ile194 on TM2, and Lys12 is close to ECL2 and/or proximal segments of TM4 and/or TM5 (Fig. 3). The proximity of Ser2 with TM7 and Gln3 with TM2 is consistent with the potential helix-helix interface between TM2 and TM7 of PTH1R. Accordingly, the high level of amino acid identity of the predicted 7TM helices of family B 7TM GPCRs probably reflects an arrangement into a conserved helical bundle structure in which TM2 is close to TM7. Proximity of TM2 and TM7 is consistent with a rhodopsin-like arrangement of the 7TM helices, although the orientation may be either clockwise or counterclockwise (34).
|
The position of ECL2 relative to the extracellular end of TM3 is probably constrained by a conserved disulfide bond of many family A and B 7TM GPCRs, and ECL2 is involved in peptide-agonist binding of family A 7TM GPCRs (3438). ECL2 of the secretin and VPAC2 receptors has been implicated in ligand binding, and specifically four residues in the N-terminal half of ECL2 contribute significantly to the secretin/VIP selectivity profile of the secretin receptor (17, 20). On the basis of the results presented here, we will attempt to identity the specific residue(s) in ECL2, TM4, and/or TM5 of GluR that determines specificity for Lys12 of glucagon.
![]() |
FOOTNOTES |
---|
** To whom correspondence should be addressed: Novo Nordisk, Molecular Pharmacology, Novo Nordisk Park C9 S.27, DK-2760 Maaloev, Denmark. Tel.: 45-44434545; Fax: 45-44434587; E-mail: bsw{at}novonordisk.com.
1 The abbreviations used are: GLP, glucagon-like peptide; GluR, glucagon
receptor(s); GLP-1R, GLP-1 receptor; TM, transmembrane; GPCR, G
protein-coupled receptor; VIP, vasoactive intestinal polypeptide; PTH,
parathyroid hormone; Nt-domain, N-terminal extracellular domain; ECL,
extracellular loop(s); ChTM2, chimeric receptor TM2; ChECL2 and -3, chimeric
receptor ECL2 and -3, respectively; PACAP, pituitary adenylate
cyclase-activating polypeptide.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|