title: Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor.
abstract: Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.
authors.affiliation: Division of Bone and Mineral Metabolism, Harvard-Thorndike and Charles A. Dana Laboratories, Harvard Institute of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Suva
authors.forename: M S
authors.initials: MS
authors.lastname: Flannery
authors.forename: M P
authors.initials: MP
authors.lastname: Caulfield
authors.forename: D M
authors.initials: DM
authors.lastname: Findlay
authors.forename: H
authors.initials: H
authors.lastname: Jüppner
authors.forename: S R
authors.initials: SR
authors.lastname: Goldring
authors.forename: M
authors.initials: M
authors.lastname: Rosenblatt
authors.forename: M
authors.initials: M
authors.lastname: Chorev
mesh_terms: D000595:Amino Acid Sequence; D001577:Benzophenones / Q000138:chemical synthesis / Q000378:metabolism*; D001667:Binding, Competitive; D002116:Calcitonin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D015195:Drug Design; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels / Q000378:metabolism*; D018003:Receptors, Calcitonin / Q000032:analysis* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D001577:Benzophenones; D019920:Photoaffinity Labels; D018003:Receptors, Calcitonin; C047723:benzophenone; D002116:Calcitonin; D000242:Cyclic AMP