An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

Abstract

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

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

“…Even though the polar core is conserved in all members of class B GPCRs, we have noted previously that there is a differential requirement of the receptor ECD for activation of class B GPCRs (16,24). In the case of GCGR and GLP-1R, the presence of the ECD is required for ligand-mediated activation of the receptor, suggesting an ECD-TMD contact during the receptor activation process.…”

“…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.…”

“…The first example was the activation of GLP-1R by small molecule electrophiles such as BETP, which modifies Cys-347 near TM6 (23). C347R/K mutations in GLP-1R allow activation of GLP-1R by fusion of a nonspecific 5-residue linker that is devoid of any GLP-1 sequence (24). The hydrophobic lock residue Phe-345 in GCGR is analogous to Cys-347 in GLP-1R.…”

“…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.…”

“…The AD293 or HTL (18,24) cell lines were routinely grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, Life Technologies) in a humidified chamber supplied with 5% CO 2 and 37°C constant temperature. Cells reaching 80 -90% confluence were detached by trypsin and reseeded by 4 -6-fold dilution in fresh medium for the assays.…”