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

• Published in: Gene therapy Vol. 8; no. 19; pp. 1490 - 1498
• Main Authors: LIANG, Q, SATYAMURTHY, N, BARRIO, J. R, TOYOKUNI, T, PHELPS, M. P, GAMBHIR, S. S, HERSCHMAN, H. R
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01.10.2001
• Subjects: Adenoviridae - genetics; Adenovirus; Adenoviruses; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Biological and medical sciences; Biotechnology; Cell Line; Colforsin - pharmacology; Cyclic AMP; Cyclic AMP - metabolism; Diverse techniques; Dopamine; Dopamine - pharmacology; Dopamine D2 receptors; Forskolin; Fundamental and applied biological sciences. Psychology; Gene expression; Gene therapy; Genes, Reporter; Genetic Vectors - administration & dosage; Guanine nucleotide-binding protein; Health. Pharmaceutical industry; Industrial applications and implications. Economical aspects; Injection; Injections, Intravenous; Intravenous administration; Ligands; Liver - metabolism; Medical sciences; Mice; Molecular and cellular biology; Mutants; Mutation; Point Mutation; Positron emission tomography; Protein Binding; Rats; Receptors, Dopamine D2 - genetics; Reporter gene; Signal Transduction; Spiperone; Spiperone - metabolism; Tomography, Emission-Computed; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Tumor Cells, Cultured; Tumors; Xenografts; Adenoviridae; Dopamine receptor; Rodentia; Gene expression; Heterograft; Genetic transfer; Virus; Vertebrata; Mammalia; Reporter gene; D2 Dopamine receptor; Mouse; Imaging; Mutation; Gene therapy; Positron emission tomography; Quantitative analysis
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3301542

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.