Abstract
We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.
Highlights
A molecular understanding of agonist-binding determinants provides important insights that may be useful in the development of drugs acting at the receptor of interest
We have recently been successful in using this approach to identify the site of contact between a photolabile nitrophenylalanine positioned at the carboxyl terminus of cholecystokinin (CCK)1 and its receptor [1]
Digestion of the earlier eluting peak yielded a product that comigrated with a parabenzoylphenylalanine standard, while the specificity of this protease did not permit the release of this residue from the D-Bpa analogue
Summary
A molecular understanding of agonist-binding determinants provides important insights that may be useful in the development of drugs acting at the receptor of interest. Photoaffinity labeling has been a powerful approach to directly identify sites of interaction between a photolabile ligand and its receptor It has been limited by the low efficiency of covalent labeling and selective nature of some labeling reagents [6]. They are not quenched by water in the medium, making more reagent available to establish a covalent bond with adjacent proteins When such a residue is optimally sited within a ligand, such that it is positioned adjacent to a receptor target upon binding, the efficiencies of covalent labeling can be very high [8]. There are examples reported of efficiencies of covalent labeling of 60 –100% of bound receptor [9] Characterization of this CCK analogue demonstrated that it is a full agonist and is an effective and efficient photoprobe. Shown are means Ϯ S.E. for three assays performed in duplicate
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