Pro-Leu-Gly-NH 2 (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D 2 receptors within the CNS. However, the precise binding site of PLG is unknown. Potential photoaffinity labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity labeling ligands were designed and synthesized based upon γ-lactam PLG peptidomimetic 1. The 4-azido-benzoyl and 4-azido-2-hydroxy-benzoyl photoaffinity labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [ 3 H]N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity labeling agents are binding at the same site as PLG and PLG peptidomimetic 1.

The present study was undertaken to investigate the mechanistic role of L-prolyl-L-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D2L receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [125I]-1b to a 51 kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D2L specific antibody, in combination with autoradiography, following a two dimensional gel separation, suggested this 51 kDa protein to be the dopamine D2L receptor. Further evidence for binding of 1b to dopamine D2L was provided by samples immunoprecipitated with the D2L antibody. These samples were analyzed by western blotting in parallel with autoradiography of [125I]-1b labeled protein. Both methods revealed bands at 51 kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D2L receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D2L receptor.