title: Alanine scanning mutagenesis of the second extracellular loop of type 1 corticotropin-releasing factor receptor revealed residues critical for peptide binding.
abstract: Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
authors.forename: Kostas
authors.initials: K
authors.lastname: Gkountelias
authors.forename: Theodoros
authors.initials: T
authors.lastname: Tselios
authors.forename: Maria
authors.initials: M
authors.lastname: Venihaki
authors.forename: George
authors.initials: G
authors.lastname: Deraos
authors.forename: Iakovos
authors.initials: I
authors.lastname: Lazaridis
authors.forename: Olga
authors.initials: O
authors.lastname: Rassouli
authors.forename: Achille
authors.initials: A
authors.lastname: Gravanis
authors.forename: George
authors.initials: G
authors.lastname: Liapakis
mesh_terms: D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D036361:Peptide Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099052:CRF Receptor, Type 1
chemical_list: D029845:Amphibian Proteins; D010446:Peptide Fragments; D036361:Peptide Hormones; D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D000409:Alanine