Binding differences of human and amphibian corticotropin-releasing factor type 1 (CRF(1)) receptors: identification of amino acids mediating high-affinity astressin binding and functional antagonism.
The corticotropin-releasing factor (CRF) type 1 receptors (CRF(1)) from human (hCRF(1)) and Xenopus (xCRF(1)) differ from one another by their agonist- and antagonist-binding preference. While the agonist-binding site of the xCRF(1) receptor has been mapped, the amino acids that mediate binding of the potent peptide antagonist astressin are unknown. By constructing receptor chimeras followed by site-directed mutagenesis, the astressin-binding site of the xCRF(1) receptor was located between residues 76 and 83. This region partially overlaps with the agonist-selective domain of the xCRF(1) receptor (residues 76-89). Mutagenesis of the amphibian residues Gln(76), Gly(81) and Val(83) to the human sequence (Arg(76)Asn(81)Gly(83)) generated a receptor mutant that bound astressin with even higher affinity than the native hCRF(1) receptor. An amino acid doublet (Glu(70)Tyr(71)) that is conserved in the xCRF(1) and hCRF(2(a)) receptor after incorporation into the hCRF(1) receptor sequence was found to facilitate antagonist binding up to 15-fold higher. In agreement with the binding data, astressin was a more potent functional antagonist at receptors expressing the Glu(70)Tyr(71) motif. These data show that the agonist- and antagonist-binding sites of the hCRF(1) receptor partially overlap and that two amino acids within the N terminus of the hCRF(1) receptor negatively influence binding and functional antagonism of astressin.
F Hoffmann-La Roche AG, Grenzacher Strasse 124, 4005 Basel, Switzerland. fdautzen@prdbe.jnj.com
Frank M
FM
Dautzenberg
Sandra
S
Wille
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D029845:Amphibian Proteins; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D036361:Peptide Hormones; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011869:Radioligand Assay; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D014162:Transfection; D054832:Urocortins; D014981:Xenopus; D000099052:CRF Receptor, Type 1
D029845:Amphibian Proteins; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D054832:Urocortins; C104416:astressin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000099052:CRF Receptor, Type 1; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; C045089:corticotropin releasing hormone (9-41); D000242:Cyclic AMP