Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH2 to the dopamine D2L receptor: Evidence for the allosteric modulation of the dopamine receptor

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.