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

Abstract

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...

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

“…2a). Consistent with the previous report (16), in the absence of any ligand, WT GCGR has a very low basal activity, and addition of a membrane-tethered GCG (GCG-M) stimulated an approximately 30-fold increase in cAMP activity (Fig. 2b), which was approximately the same level of activation by exogenous GCG at saturated concentration (1 M GCG in Fig.…”

“…Ligand binding to the GCGR ECD rearranges the ECD to a "stand up" position and releases the ECD repression of the TMD, which then results in receptor activation. Although the stand up activation model fits well with the general framework of the two-domain mechanism for ligand binding and activation of class B GPCR, it cannot explain the requirement of the ECD in activation of GCGR and GLP-1R (16). In these two receptors, we have shown that their ECDs play a much more direct role in the receptor activation in addition to their role in ligand binding (16).…”

“…1). Using AD293 cells expressing exogenous GCGR as our model system (16,24), we determined cAMP-dependent reporter gene activity as measured for the basal and activated activity of GCGR/G s signaling in the absence or presence of GCG hormonal peptide. In these experiments, we fused glucagon (residues 1-29) to a long flexible linker and the single membrane-spanning helix of CD8 (25) and co-expressed these chimeric peptides with wild-type (WT) and mutated full-length GCGRs (Fig.…”

“…Although the stand up activation model fits well with the general framework of the two-domain mechanism for ligand binding and activation of class B GPCR, it cannot explain the requirement of the ECD in activation of GCGR and GLP-1R (16). In these two receptors, we have shown that their ECDs play a much more direct role in the receptor activation in addition to their role in ligand binding (16). However, the absence of a fully activated class B GPCR structure has limited our mechanistic understanding of ligand binding and receptor activation for this important family of receptors.…”

Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

“…Preliminary studies have proposed that GPCR ECRs regulate receptor functions, likely including G-protein signaling, on binding to extracellular ligands (9,(23)(24)(25)(26)(27)(28)(29). Two complementary models for ligand-induced aGPCR activation have been proposed (Fig.…”