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 (CRF1R), 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.
Highlights
In the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the extracellular domain (ECD) is required for signaling even when the hormone is covalently linked to the transmembrane domain (TMD)
In this report we demonstrate that the ECDs of some class B G protein-coupled receptors (GPCRs), such as CRF1R, PAC1R, and PTH1R, solely function as affinity traps, and their requirement can be bypassed by mass action effects and hormone tethering, consistent with the prevailing model of peptide binding and activation of Class B GPCRs
Class B GPCRs Differ in Their ECD Requirements for Signaling—To examine the requirement of the GCGR ECD for signaling, we transiently transfected four different FLAGtagged constructs into HEK293 cells: the full-length receptor, TMD, TMD-interacting N terminus of glucagon [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15] fused to the TMD, and a fusion in which glucagon [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15] and the TMD are separated by a thermostabilized cytochrome b562 variant, BRIL, which has been used for facilitating GPCR crystallization [16] (Fig. 1, B–D)
Summary
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. 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].
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