Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Abstract

a b s t r a c tGlucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO 2 -biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short ␤ stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.

Structure of the full-length glucagon class B G-protein-coupled receptor

“…3b). The observed conformation of ECL1 is supported by the previous study using a cysteine accessibility method that suggested that this extracellular loop of GCGR is in an extended conformation, probably a β-sheet 18 . Furthermore, it was proposed that ECL1 forms a compact structure or interacts with other parts of the receptor 18 .…”

“…The observed conformation of ECL1 is supported by the previous study using a cysteine accessibility method that suggested that this extracellular loop of GCGR is in an extended conformation, probably a β-sheet 18 . Furthermore, it was proposed that ECL1 forms a compact structure or interacts with other parts of the receptor 18 . This agrees with the GCGR-FL crystal structure in which ECL1 of the receptor runs in parallel with the stalk and its backbone from L210–T214 forms hydrogen bonds with the main chain of the residues E126–V130 on the stalk, forming a compact β-sheet structure (Fig.…”

“…This aligns with the HDX studies demonstrating that regions of both the stalk and ECL1 displayed increased protection in the peptide-bound GCGR compared with the small-molecule antagonist-bound receptor, suggesting that these two regions adopt variable conformations when different ligands bind to the receptor 7 . A previous cysteine accessibility study found that accessibility of the mutant L198 2.71b C on the extracellular side of helix II was low in the absence of ligand but increased upon glucagon binding 18 , indicating that the residue L198 2.71b , which was reported to be involved in glucagon binding 8 , might be buried by other residues of the receptor in the inactive state and exposed to an aqueous environment in active conformation. In the inactive GCGR-FL structure, L198 2.71b is right beneath ECL1 and the stalk, which may provide a ‘shelter’ for this residue in the inactive state and change conformation to facilitate peptide ligand binding in the active state.…”

“…GCGR and some other class B GPCRs, including GLP-1R, parathyroid hormone receptor (PTHR) and gastric inhibitory polypeptide receptor (GIPR), contain a long ECL1 with 11–26 residues, as compared with 4–6 residues in other class B and most class A GPCRs. Previous studies reported that the ECL1 of GCGR might participate in binding to its endogenous ligand glucagon and regulating receptor activity 17,18 . However, the regulation mechanism of GCGR activation by its ECL1 remained unclear due in part to the absence of the conformational information of ECL1 in the previously solved crystal structures of GCGR-TMD.…”

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

“…Induced conformational changes in the ECL2 have been documented in several GPCRs, including the D2 dopamine receptor and C5A complement receptor (44,45) upon ligand occupancy of the orthosteric pocket in the TM domain. Long range conformational changes associated with ligand binding have been shown in ECL1 of glucagon receptor (46); the N terminus, ECL1, and extracellular ends of all TM helices of Saccharomyces cerevisiae G protein-coupled receptor Ste2p are known (47)(48)(49). These observations suggest that a mutation in a particular domain of the receptor causes a local structural perturbation that is eventually transmitted to other domains, leading to a global consequence in terms of affinity toward ligands and G protein.…”