Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue.
Receptor recognition by the Asp(3) residues of vasoactive intestinal peptide and secretin requires the presence of a lysine residue close to the second transmembrane helix (TM2)/first extracellular loop junction and an ionic bond with an arginine residue in TM2. We tested whether the glucagon Gln(3) residue recognizes the equivalent positions in its receptor. Our data revealed that the binding and functional properties of the wild-type glucagon receptor and the K188R mutant were not significantly different, whereas all agonists had markedly lower potencies and affinities at the I195K mutated receptor. In contrast, glucagon was less potent and the Asp(3)-, Asn(3)- and Glu(3)-glucagon mutants were more potent and efficient at the double-mutated K188R/I195K receptor. Furthermore, these alterations were selective for position 3 of glucagon, as shown by the functional properties of the mutant Glu(9)- and Lys(15)-glucagon. Our results suggest that although the Gln(3) residue of glucagon did not interact with the equivalent binding pocket as the Asp(3) residue of vasoactive intestinal peptide or secretin, the Asp(3)-glucagon analogue was able to interact with position 188 of the K188R/I195K glucagon receptor. Nevertheless, the Gln(3) side chain of glucagon probably binds very close to this region in the wild-type receptor.
Perret
Jason
J
Department of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Bât G/E, CP 611, 808 Route de Lennik, B-1070 Bruxelles, Belgium.
Van Craenenbroeck
Mélanie
M
Langer
Ingrid
I
Vertongen
Pascale
P
Gregoire
Françoise
F
Robberecht
Patrick
P
Waelbroeck
Magali
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004252:DNA Mutational Analysis; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000737:chemistry* / Q000235:genetics; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; C029937:secretin receptor