Mutational Analysis and Molecular Modeling of the Nonapeptide Hormone Binding Domains of the [Arg8]vasotocin Receptor

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 14; pp. 6907 - 6912
• Main Authors: Hausmann, Holger, Richters, Anke, Kreienkamp, Hans-Jürgen, Meyerhof, Wolfgang, Mattes, Henri, Lederis, Karl, Zwiers, Henk, Richter, Dietmar
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 09.07.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Agonists; Amino Acid Sequence; Amino acids; Animals; Binding Sites; Biochemistry; Cell Line; Cell Membrane - metabolism; Cell membranes; Chimeras; Complementary DNA; DNA Mutational Analysis; Female; Fish; Fishes; Hormones; Humans; Inhibitory concentration 50; Kidney; Kidney cells; Kidneys; Kinetics; Ligands; Membranes; Models, Molecular; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oocytes; Oocytes - physiology; Peptides; Polymerase Chain Reaction; Protein Structure, Secondary; Rats; Receptors; Receptors, Vasopressin - biosynthesis; Receptors, Vasopressin - chemistry; Receptors, Vasopressin - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Transfection; Tritium - metabolism; Vasotocin - metabolism; Xenopus laevis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.14.6907

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H) Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.