Isolation and structure-function studies of a glucagon-like peptide 1 receptor from goldfish Carassius auratus: identification of three charged residues in extracellular domains critical for receptor function.

A better understanding of the molecular mechanism of ligand-receptor interaction of glucagon-like peptide 1 (GLP-1) receptors (GLP-1Rs) is useful for the design of potent GLP-1 analogs that could potentially be used as a treatment for diabetic patients. Changes in the ligand and receptor sequences during evolution provide invaluable clues to evaluate the functional motifs of the receptor that are responsible for ligand interaction. For these reasons, in the present study, we have isolated and functionally characterized a GLP-1R from goldfish. Its amino acid sequence shows 50.8% and 52.3% identity with the human glucagon (hGLU) and GLP-1Rs, respectively, and 84.1% with the zebrafish GLP-1R (the only other GLP-1R isolated from teleost fish). Peptides that are structurally different from goldfish (gf)GLP-1, such as gfGLU and hGLU and human GLP-1 (7-36)amide, are also capable of stimulating this receptor, albeit with lower potencies than gfGLP-1. gfGLP-1 stimulates the formation of cAMP through the recombinant gfGLP-1R with EC(50) = 0.18 nM, whereas EC(50) values for gfGLU, human GLP-1 (7-36)amide, and hGLU are 0.53 nM, 0.9 nM, and 1.2 nM, respectively. These results indicate that the gfGLP-1R is structurally more flexible than its mammalian counterpart and that its binding pocket can accommodate a wider spectrum of peptide ligands. Previous studies demonstrated that the charged residues in the extracellular domains of mammalian GLP-1R, particularly those found in the N-terminal domain and the first exoloop, are important for ligand binding. We investigated the roles of the conserved charged residues in the function of the gfGLP-1R. Eleven mutant receptors were constructed, and the effects of mutations were determined by functional assays. Our results demonstrated that three charged residues (D(113), R(197), and D(205)) present in the extracellular domains are critical for receptor function.