Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gone. D2R reporter gen e expression has been non-Invasively, repetitively and quantitatively image d by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2 '-[F-18]-fiuoroethyl)-spiperone, FESP) and su bsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporte r gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs t hat still bind [H-3]spiperone in transfected cells. The D2R80A mutation com pletely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R8 0A expression plasmid and in cells infected with replication-defective aden ovirus expressing D2R80A. The D2R194A mutation substantially reduces, but d oes, not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated eq uivalent [H-3]spiperone binding activity. Moreover, hepatic FESP sequestrat ion is equivalent, following intravenous injection of adenoviruses expressi ng D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP lev els following ligand binding, has full capability as a PET reporter gene.