Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH 2 to the dopamine D 2L receptor: Evidence for the allosteric modulation of the dopamine receptor

Abstract

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 ∼ 51 kDa 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 ∼ 51 kDa 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 ∼ 51 kDa. 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.