Identification and characterization of two nuclear factor-kappaB sites in the regulatory region of the dopamine D2 receptor.

Regulation of D2 receptor (D2R) expression is crucial in the function of dopaminergic systems. Because alterations of D2R expression may contribute to the development of different disorders, it is important to elucidate the mechanisms regulating D2R gene transcription. We report the characterization of two putative nuclear factor-kappaB (NF-kappaB) motifs, referred to as D2-kappaB sites, in the human D2R promoter, and demonstrate that they bind NF-kappaB subunits and stimulate D2R promoter activity. D2-kappaB sites show different degrees of conservation and specificity, when compared with canonical kB sites. The D2-kappaB1 site (from -407 to -398) is highly conserved and binds p50/p65 and p50/c-Rel complexes, whereas D2-kappaB2 (from -513 to -504) is more degenerated and only binds p50/p65 heterodimers. Activation of D2-kappaB sites in COS-7 cells expressing a luciferase reporter vector containing the D2R promoter resulted in increased transcriptional activity. Site-directed mutagenesis of each D2-kappaB site differentially modified D2R promoter activity. In particular, mutation of the D2-kappaB1 motif did not affect D2R promoter response to p50/c-Rel complexes, whereas inactivation of the D2-kappaB2 site decreased it. Mutations of either D2-kappaB1 or D2-kappaB2 sites attenuated the D2R promoter transcriptional efficiency induced by p50/p65 complexes. Thus, D2R transcription mediated by p50/c-Rel is supported mainly by the D2-kappaB2 site, whereas both sites are necessary to support the full transcriptional activity mediated by p50/p65 complexes. A correlation was found between NF-kappaB activity and D2R expression in the pituitary and pituitary-derived cells but not in the striatum, suggesting that NF-kappaB regulation of D2R expression could be a pituitary-specific mechanism.

Loss of synaptic D1 dopamine/N-methyl-D-aspartate glutamate receptor complexes in L-DOPA-induced dyskinesia in the rat.

Glutamate-mediated mechanisms are related to the motor complications of L-DOPA therapy in Parkinson's disease (PD). In striatal postsynaptic densities (PSD), the dopamine D1 receptor (D1R) is part of an oligomeric complex with the glutamate N-methyl-D-aspartate receptor (NMDAR), determining the strength of corticostriatal transmission. We studied D1R/NMDAR complex alterations induced by L-DOPA in the 6-hydroxydopamine-lesioned rat model of PD. L-DOPA-treated hemiparkinsonian rats were determined to be dyskinetic or nondyskinetic based on behavioral testing. D1R/NMDAR assemblies containing NR1-C2 and NR2B subunits were decreased in the PSD of lesioned striatum. Short-term L-DOPA administration improved akinesia and restored the synaptic abundance of D1R, NR1-C2 and NR2B. Prolonged L-DOPA treatment also normalized synaptic D1R/NMDAR complexes in nondyskinetic rats, but remarkably reduced them in the dyskinetic group without changing their interaction. This decrease involved NR1-C2, NR1-C2', NR2A, and NR2B subunits. The composition of residual synaptic D1R/NMDAR complexes in dyskinetic rats may thus be different from that observed in lesioned rats, suggesting that expression of different motor dysfunctions might be related to the receptor profile at corticostriatal synapses. The levels of D1R/NMDAR complexes were unchanged in total striatal membrane proteins, suggesting that the decrease of these species in the PSD is likely to reflect an altered receptor trafficking. In human embryonic kidney 293 cells expressing the D1R/NMDAR, complex costimulation of both D1R and NMDAR, but not individual receptor activation, promoted internalization, suggesting that development of dyskinesias might be related to agonist-mediated down-regulation of the D1R/NMDAR complex at corticostriatal synapses.