Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH₂ to the dopamine D_2L 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 D_2L 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~51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D_2L-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this~51kDa protein to be the dopamine D_2L receptor. Further evidence for binding of 1b to dopamine D_2L was provided by samples immunoprecipitated with the D_2L antibody. These samples were analyzed by western blotting in parallel with autoradiography of [¹²⁵I]-1b labeled protein. Both methods revealed bands at~51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D_2L 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 D_2L receptor.