Abstract
The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.
Highlights
Grant 3.4541.03 and by an “Action de Recherche Concertee” from the Communaute Francaise de Belgique. 1 Both authors contributed to this work. 2 Present address: Human Metabolism, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield ST10 2RX, UK. 3 To whom correspondence should be addressed: Laboratoire de Chimie Biologique et de la Nutrition, Facultede Medecine, 808 Route de Lennik CP611, 1070 Bruxelles, Belgium
Except for two cysteine residues in the first and second extracellular loops, none of the signature amino acids defining family A G protein-coupled receptors are identified in family B receptors
The three-dimensional structures of the N-terminal domain of the CRF2, PAC1, glucagon-like peptide 1 (GLP-1), and gastrointestinal insulinotropic peptide (GIP) receptors and of the CRF1 or parathyroid hormone receptor N-terminal region fused with the maltose-binding protein, in complex with their respective agonist or antagonist ligands, have been determined by NMR or x-ray diffraction studies (6 –12)
Summary
Modeling—The positions of the signal peptide (residues 1–26) and of the transmembrane helices, intracellular (IC) and extracellular (EC) regions were predicted using the SignalP 3.0 server and the Predict Protein server, respectively [17, 18]. Western Blots—Fifty g of membrane proteins were solubilized at room temperature in Laemmli sample buffer, resolved by SDS-PAGE using a 10% gel, and electrotransferred on a PVDF membrane This membrane was blocked for 1 h in PBS enriched with 5% dry milk powder (Nestle) and 1‰ Tween 20, incubated for 1 h in the presence of a rabbit antiserum raised against a peptide corresponding to the C-terminal glucagon receptor sequence (SAETPLAGGLPRLAESPF (1:25,000 in the blocking buffer)), and washed in PBS-Tween (1‰). Precipitation by ethanol (9 volumes, overnight at Ϫ20 °C) or Biotinylation and Biotin Pullout—Intact cells (or mem- TCA (10% at 4 °C) as well as solubilization in SDS resulted in branes) were treated for 10 min at 25 °C in 120 l of PBS with cumulative decreases in the receptor immunodetection; it was MTSEA-biotin (1 mM, 1% DMSO) or maleimide-PEO2 biotin (0.4 mM) and washed four times at 4 °C, resuspended in ice-cold 1 mM NaHCO3 enriched with CompleteTM, frozen in liquid nitrogen, and stored at Ϫ80 °C until use. The double mutants (charge switch) D63K/K98D and compared with wild type by one-way analysis of variance, using the D63R/R116D did not restore the glucagon potency
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