Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket

Mann, Richard P.; Al-Sabah, Suleiman; Maturana, Rakel López de; Sinfield, John K.; Donnelly, Dan

doi:10.1016/j.peptides.2010.09.015

Cited by 19 publications

(25 citation statements)

References 36 publications

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“…Other connections that may support EC2 include an aromatic-stacking interaction between W284 of EC2 and F230 on TM3, and hydrogen bonds between K288 of EC2 and P283 of TM4 and between Y289 of EC2 and Y305 of TM5. Mutation of W284 to alanine is also likely to change EC2 conformation and the preferential effect on GLP-1 affinity again implicates this extracellular loop in GLP-1 binding, consistent with other recent work implicating this loop in both binding and receptor activation (46). Further, mutation of K288 to alanine reduces GLP-1 affinity (18), and our model indicates that this may result from the importance of this residue in the conformation of EC2 rather than the result of a direct interaction with the ligand.…”

Section: Discussionsupporting

confidence: 86%

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Coopman

Wallis

Robb

et al. 2011

Molecular Endocrinology

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The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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Section: Discussionsupporting

confidence: 86%

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Coopman

Wallis

Robb

et al. 2011

Molecular Endocrinology

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“…This receptor residue is adjacent to the functionally important conserved Cys 296 that forms a disulfide bond with Cys 226 within ECL1 (55). The importance of ECL2 in ligand binding has also been suggested in mutagenesis and chimeric receptor studies for several family B GPCRs, including receptors for GLP1 (56), CRF 1 (57), CRF 2 (58), secretin (23), PTH 1 (59), PTH 2 (60), and glucagon (61).…”

Section: Discussionmentioning

confidence: 97%

Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

Miller

Chen

Lam

et al. 2011

Journal of Biological Chemistry

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The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino-and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-L-phenylalanine into positions 16 and 20 of GLP1(7-36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu 141 above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp 297 within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region. G protein-coupled receptors (GPCRs)4 comprise a large family of plasma membrane receptors, binding molecules outside the cell that initiate intracellular signal transduction pathways and cellular responses. These molecules are targets for more than one-third of approved drugs. Family B GPCRs represent a relatively small group of receptors that nonetheless include important potential drug targets. Among these are receptors for secretin, vasoactive intestinal polypeptide, corticotropinreleasing factor (CRF), glucagon, glucagon-like peptide 1 (GLP1), calcitonin, and parathyroid hormone (PTH) (1).The GLP1 receptor is an important drug target within family B GPCRs. Its natural ligand, GLP1, has multiple antidiabetic actions, including promotion of insulin secretion, the preservation of ␤-cell mass, and the ability to lower body weight. A series of drugs have been developed and introduced that target this receptor for the treatment of type 2 diabetes mellitus (2). Like other natural ligands of this family, GLP1 is a moderately large and flexible peptide with a diffuse pharmacophore extending throughout its entire length, providing challenge to our understanding of its interaction with its receptor.Family B GPCRs all have a long and structurally characteristic extracellular amino-terminal domain stabilized by three pairs of disulfide bonds linking six conserved cysteine residues. This domain functions as the pred...

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“…For Cys-296, a structural role is clearly evident with this amino acid forming a disulfide bond with Cys-226 at the top of TM3; this structural motif is highly conserved across the superfamily of GPCRs and is evident in solved crystal structures for family A GPCRs (57-61, 75, 76). In the rat GLP-1R, double mutation of Cys-226 and Cys-296 to alanine restored the loss of GLP-1(7-36)-NH 2 binding affinity and cAMP signaling seen with individual mutation of these residues, indicating that the disulfide link itself is not required for efficient activation of the receptor (77).…”

Section: Journal Of Biological Chemistrymentioning

confidence: 98%

“…Curiously, double mutation of Tyr-305 and Trp-306 to alanine in the rat GLP-1R resulted in a population of receptors that were cell surface-expressed and responded to peptide agonists, albeit with marked effects on potency (77), suggesting that either interspecies differences in receptor sequence provide for greater stability of rat receptor structure or that the additional mutation compensated for some of the detrimental interactions arising from individual mutation of Trp-306.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Has a Critical Role in GLP-1 Peptide Binding and Receptor Activation

Koole

Wootten

Simms

et al. 2012

Journal of Biological Chemistry

121

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Background: The ECL2 of family B GPCRs has been suggested to contribute to biological activity. Results: Mutation of most ECL2 residues to alanine results in changes in binding and/or efficacy of GLP-1 peptide agonists. Conclusion: The ECL2 of the GLP-1R is critical for GLP-1 peptide-mediated receptor activation and selective signaling. Significance: This work reveals broad significance for ECL2 in maintaining receptor conformations driving selective signaling.

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Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket

Cited by 19 publications

References 36 publications

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Has a Critical Role in GLP-1 Peptide Binding and Receptor Activation

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