Three Distinct Epitopes on the Extracellular Face of the Glucagon Receptor Determine Specificity for the Glucagon Amino Terminus

Runge, Steffen; Gram, Christian; Bräuner‐Osborne, Hans; Madsen, Kjeld; Knudsen, Lotte Bjerre; Wulff, Birgitte S.

doi:10.1074/jbc.m301085200

Cited by 70 publications

(78 citation statements)

References 39 publications

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“…The C-terminal fragment of PTH (residues 15-34) binds to the N-terminal ECD of the receptor to confer high affinity and specificity to the receptor (28,29). This ''two-domain'' model of PTH binding and activation is further supported by studies with chimeric ligands and receptors (30)(31)(32) and has since been demonstrated for other class B GPCR molecules (33)(34)(35).…”

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confidence: 71%

“…Crystals of the purified MBP-PTH1R-ECD bound to a synthetic PTH fragment (residues [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] were grown in hanging drops containing a reservoir solution of 100 mM sodium cacodylate (pH 6.5) and 30% (vol/vol) polypropylene glycol P400 (PPG P400). Diffraction data were collected at 21-ID-D (LS-CAT) of the Advanced Photon Source at Argonne National Laboratory (Argonne, IL).…”

Section: Methodsmentioning

confidence: 99%

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Molecular recognition of parathyroid hormone by its G protein-coupled receptor

Pioszak

2008

Proc. Natl. Acad. Sci. U.S.A.

238

313

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Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-Å structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer ␣-␤-␤␣ fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.calcitonin ͉ crystal structure ͉ glucagon ͉ maltose-binding protein ͉ osteoporosis

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mentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Molecular recognition of parathyroid hormone by its G protein-coupled receptor

Pioszak

2008

Proc. Natl. Acad. Sci. U.S.A.

238

313

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“…We assumed that K187 and I194, that affect the receptor specificity for glucagon Gln3 [24,30], are oriented inside the binding pocket. As shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…These receptors possess a large N-terminal receptor domain with 6 conserved cysteines forming 3 essential disulfide bridges, followed by a serpentine arrangement of the polypeptide involving seven transmembrane helical domains. Investigation of chimeric receptor activation by hybrid peptides showed that, like Glucagon Like Peptide-1 (GLP-1) and Gastric Inhibitory Peptide (GIP) [22,31,42], the glucagon carboxyl-terminal half is anchored on the receptor extracellular Nterminal domain, and the receptor is activated by the interaction of the amino-terminal half of the peptide with the transmembrane domain [30,32,39]. This conclusion has been supported by several other studies: glutamate 68 in the GLP-1 receptor N-terminal domain (replaced by K64 in the glucagon receptor) reduces the GLP-1 receptor affinity for glucagon by charge repulsion with its' C-terminal carboxyl [4,8]; an aspartate (D385, replaced by E387 in the GLP-1 receptor) in TM7 is responsible for preferential recognition of the glucagon S2 over GLP-1 A2; K187 and I194 side chains in TM2 participate in the preference for glucagon Q3 [24,30] over GLP-1 E3; and unidentified side chains in ECL2, for glucagon K12 [30].…”

Section: Abbreviationsmentioning

confidence: 99%

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

2011

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a b s t r a c tGlucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO 2 -biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short ␤ stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.

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“…However, these receptors may also have distinct structures formed by ortholog-specific residues in the core domain that interact with ortholog-specific residues in the corresponding peptide. For example, in addition to the highly common residues Gly 4 , Phe 6 , The 7 , and Asp/Glu 9 , glucagon-specific residues, Ser 2 , Gln 3 , Tyr 10 , and Lys 12 , are also important for receptor binding and activation (60,61). Indeed, a recent glucagon-docked GCGR modeling based on the antagonist-bound GCGR crystal structure revealed putative interactions of these glucagon-specific residues with GCGR (38).…”

mentioning

confidence: 99%

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

Moon

Lee

Park

et al. 2015

Journal of Biological Chemistry

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Background:Little is known about the interaction between GLP-1 and the heptahelical core domain of GLP1R. Results: GLP-1 Asp 9 and Gly 4 interact with the evolutionarily conserved residues in extracellular loop 3. Conclusion: Ligand binding pocket formed by evolutionarily conserved residues in the GLP1R core domain. Significance: This study highlights the mechanism underlying high affinity interaction between GLP-1 and the binding pocket of the receptor.

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Three Distinct Epitopes on the Extracellular Face of the Glucagon Receptor Determine Specificity for the Glucagon Amino Terminus

Cited by 70 publications

References 39 publications

Molecular recognition of parathyroid hormone by its G protein-coupled receptor

Molecular recognition of parathyroid hormone by its G protein-coupled receptor

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

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