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

• Published in: Peptides (New York, N.Y. : 1980) Vol. 32; no. 8; pp. 1593 - 1599
• Main Authors: Roberts, David J., Vertongen, Pascale, Waelbroeck, Magali
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01-08-2011; Elsevier
• Subjects: Amino Acid Sequence; Binding Sites; Biological and medical sciences; biotin; Cells, Cultured; cysteine; Cysteine - genetics; Cysteine - metabolism; Fundamental and applied biological sciences. Psychology; Glucagon; Glucagon - metabolism; Glucagon binding site; Glucagon receptor; glucagon receptors; Humans; hydrophilicity; hyperglycemia; hypoglycemia; Mesylates - pharmacology; Molecular Sequence Data; Mutagenesis (site directed); mutation; patients; Receptors, Glucagon - genetics; Receptors, Glucagon - metabolism; Substituted Cysteine Accessibility Method (SCAM); Transfection; Vertebrates: endocrinology; Glucagon receptor; pEC50; Glucagon; DMEM medium; ECL; HEK; Maleimide-PEO2-biotin; Mutagenesis (site directed); EC50; TM; Substituted Cysteine Accessibility Method (SCAM); SCAM; Glucagon binding site; MTSET; Pancreatic hormone; Thiol; Cysteine; Peptide hormone; Binding site; Extracellular; Sulfur containing aminoacid; Mutagenesis; Biological receptor; Substituted Cysteine Accessibility Method
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2011.06.009

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► SCAM of the first glucagon receptor extracellular loop (197–224). ► TM2 continues to position 202, TM3 from 215 ► Most side chains important for active glucagon conformation. ► S217 and D218 contact glucagon, V221 very close to the binding site. ► Receptor activation increases the accessibility of L198C mutant cysteine.
Glucagon 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-PEO2-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 β 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.