DESIGN, SYNTHESIS AND UTILITY OF NOVEL BENZOPHENONE-CONTAINING CALCITONIN ANALOGS FOR PHOTOAFFINITY-LABELING THE CALCITONIN RECEPTOR

Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane recep tor (CTR). In this study, we report the design, synthesis and characte rization of four potent bioactive and photoreactive CT analogs, each o f which contains a single benzophenone moiety inserted at different an d discrete locations within the CT molecule. Replacement of all Lys re sidues in salmon CT (sCT) with Arg, followed by replacement of hydroph obic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilo n-pBz(2))] was found to preserve high biological activity. We substitu ted Val(8), Leu(16) and Leu(19) by Lys(epsilon-pBz(2)), and acylated t he N-terminus by a pBz(2) moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT seque nce. With both transfected and endogenous CTRs from several species, a ll four benzophenone-containing analogs were shown to be virtually ind istinguishable from the parent sCT analog in both receptor binding pro perties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog {[Arg(11,1 8),Lys(19)(epsilon-pBz(2))]sCT (K19)} covalently labels a membrane com ponent of approximately 70 kDa. Receptor crosslinking is inhibited spe cifically in the presence of excess sCT. We also examined the interact ion of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation iden tical with native CTR. Both K19 and another bioactive analog {[Arg(11, 18),Lys(8)(epsilon-pBz(2))]sCT (K8)} specifically photoaffinity cross- link to the HA-CTR. These benzophenone-containing CT analogs should fa cilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the a nalysis of photochemically cross-linked conjugates.