Modulation of Agonist Binding to Human Dopamine Receptor Subtypes by l-Prolyl-l-leucyl-glycinamide and a Peptidomimetic Analog

Abstract

The present study was undertaken to investigate the role of the hypothalamic tripeptide l-prolyl-l-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [³H]N-propylnorapomorphine (NPA) and [³H]quinpirole binding in a dose-dependent manner to the DA D_2L,D_2S, and D₄ receptors. However, agonist binding to the D₁ and D₃ receptors and antagonist binding to the D_2L receptors by PLG were not significantly affected. Scatchard analysis of [³H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D_2L, D_2S, and D₄ receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D_2L receptor and attenuated guanosine 5′-(β,γ-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D_2L receptor. Furthermore, direct NPA binding with D_2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D_2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D_2S, D_2L, and D₄ receptors in an allosteric manner and that the coupling of D₂ receptors to the G protein is essential for this modulation to occur.