Design, Synthesis and Utility of Novel Benzophenone-Containing Calcitonin Analogs for Photoaffinity Labeling the Calcitonin Receptor
- Larry J. Suva1,
- Merrilee S. Flannery2,
- Michael P. Caulfield4,
- David M. Findlay5,
- Harald Jüppner3,
- Steven R. Goldring2,
- Michael Rosenblatt1 and
- Michael Chorev1
- 1Division 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 (L.J.S, M.R., M.C.), 2Arthritis (M.F., S.R.G.) and 3Endocrine Units (H.J.), Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02114, 4Nichols Institute Laboratories, San Juan Capistrano, CA 92690 (M.P.C.), 5St. Vincent’s Institute of Medical Research, Fitzory 3065, Australia (D.M.F.)
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(ε-p-benzoylbenzoyl) residue [Lys(ε-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16and Leu19 by Lys(ε-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(ε-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(ε-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.
Footnotes
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Send reprint requests to: Larry J. Suva, Division of Bone and Mineral Metabolism, Harvard Institutes of Medicine, Room 944, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215.
- Abbreviations:
- BIN67
- human ovarian cell line
- Boc
- t-butoxycarbonyl
- CT
- calcitonin
- CTR
- calcitonin receptor
- COS-7
- receptor negative monkey kidney cell line
- DCM
- dichloromethane
- DMF
- N,N-dimethylformamide
- DMEM. Dulbecco’s modified minimum essential medium
- e, eel
- p
- porcine
- h
- human
- FBS
- fetal bovine serum
- Fmoc
- 9-fluorenylmethoxycarbonyl
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- HF
- hydrogen fluoride
- IBMX
- 3-isobutyl-1-methylxanthine
- K8
- [Arg11,18,Lys8(ε-pBz2)]sCT
- K16
- [Arg11,18,Lys16(ε-pBz2)]sCT
- K19
- [Arg11,18,Lys19(ε-pBz2)]sCT
- LLC-PK1
- porcine kidney cell line
- Nα
- [Arg11,18,Nα(pBz2)sCT
- pMBHA
- p-methylbenzhydrylamine resin
- PBS
- phosphate-buffered saline
- PTH
- parathyroid hormone
- RP-HPLC
- reverse-phase high-performance liquid chromatography
- TFA
- trifluoroacetic acid
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- Received May 2, 1997.
- Accepted July 14, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
