Development and characterization of antibodies directed against the mouse D4 dopamine receptor

Polyclonal antibodies against the mouse D4 dopamine receptor have been developed in order to investigate the anatomical localization of this receptor in the mouse brain. Two antibodies were generated against specific peptides corresponding to predicted extracellular and intracellular regions of the D4 protein. Specificity of these antibodies was demonstrated on human embryonic kidney 293 (HEK 293) cells transfected with different dopamine receptor subtypes; immunoreactivity was detected only in cells transfected with the mouse D4 dopamine receptor cDNA. Following in vitro transcription/translation of the mouse D4 cDNA, a single protein band of 36 kDa was selectively immunoprecipitated with the anti-D4 antibodies. The antibodies also detected a single protein of 36 kDa in Western blot of HEK 293 cells transiently transfected with the mouse D4 receptor. These antibodies were able to detect the D4 receptor in several regions of the mouse brain. In the regions examined, D4 immunoreactivity was found in neurones located in layers II-VI of the frontal and piriform cortices, with the highest concentration in layer II; in scattered neurones in the caudate putamen and in larger neurones in the globus pallidus. In all experiments, both antibodies exhibit the same specificity, and all immunoreactivity could be abolished by preincubation with the corresponding peptide antigen.