A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification

Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe 6 , Thr 7 , and Leu 10 , and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys 6 -sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys 7 -sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys 10 -sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.

Section: Secretin Bindingmentioning

confidence: 90%

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Dong

Lam

Orry

et al. 2016

“…Incorporation of a cysteine at Phe-345 near the cytoplasmic side of TM6 has been shown to sensitize GCGR to the positive allosteric modulator BETP (4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine), leading to ligand-dependent positive allosteric activity similar to that of GLP-1R (23,24). In addition, Phe-345 is close to the binding site of the GCGR antagonist MK-0893 (Fig.…”

Section: Mutations At Phe-345 Are Sufficient To Induce Constitutive Rmentioning

confidence: 99%

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

Yin

Waal

et al. 2017

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ␤ 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

“…In contrast, the pyrimidine BETP (31) was not competed by exendin(9 -39) or GLP-1 and is, therefore, thought to allosterically activate GLP-1R (31)(32)(33)(34). Recent work has demonstrated that BETP functions by forming covalent adducts with Cys-347 in the intracellular loop 3 (ICL3) of GLP-1R, which may mimic a physiological covalent modification (35).…”

Section: Small Molecule Glp-1r Modulators Mimic Endogenous Peptide Homentioning

confidence: 99%

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Zhao

Yin

Yang

et al. 2016

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF 1 R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.The class B or secretin family of GPCRs consists of 15 receptors for peptide hormones that include glucagon, glucagon-like peptides, parathyroid hormone, and calcitonin (Fig. 1A). These receptors are important drug targets for many human diseases including diabetes, neurodegeneration, cardiovascular diseases, and psychiatric disorders. The full-length receptors consist of two modular domains: a globular extracellular domain (ECD) 3 defined by three conserved disulfide bonds and a TMD that contains seven transmembrane helices (1-4). The ECD is responsible for the high affinity and specificity of hormone binding, and the TMD is required for receptor activation and signal coupling to downstream G proteins and other signaling effectors (4). Peptide hormone binding is thought to proceed through fast binding of its C terminus to the ECD followed by a slower association of the peptide N terminus with the receptor TMD (5), which leads to conformational changes in the receptor TMD and the receptor activation. The activated receptors are coupled primarily to stimulatory G proteins, resulting in elevation of the intracellular cAMP level.Structures of the ECDs of class B GPCRs in complex with their peptide ligands have been determined by x-ray crystallography (1, 6 -9) and NMR (10) and have provided useful information about structural mechanisms of ligand recognition and selectivity (2, 11). Only recently, crystal structures of t...