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

Gene Therapy volume 8, pages 1490–1498 (2001)Cite this article

Abstract

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. Gene Therapy (2001) 8, 1490–1498.

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Acknowledgements

We thank Art Catapang, Raymond Basconcillo, Judy Edwards, Khoi Nguyen, Waldemar Ladno and ManKit Ho for technical assistance, Dr K Neve for advice on cyclic AMP stimulation and analysis and Duncan MacLaren for instruction in the [3H]spiperone binding assay. This work was supported by Department of Energy award DE-FC03–87ER60615, NIH award R0–1 CA84572 (HRH) and NIH award P50 CA86306 (HRH).

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Authors and Affiliations

  1. Crump Institute for Biological Imaging, UCLA School of Medicine, Los Angeles, CA, USA

    Q Liang, N Satyamurthy, JR Barrio, T Toyokuni, MP Phelps, SS Gambhir & HR Herschman

  2. UCLA/DOE Laboratory of Structural Biology and Molecular Medicine, UCLA School of Medicine, Los Angeles, CA, USA

    N Satyamurthy, JR Barrio, MP Phelps, SS Gambhir & HR Herschman

  3. Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA, USA

    Q Liang, N Satyamurthy, JR Barrio, T Toyokuni, MP Phelps, SS Gambhir & HR Herschman

  4. Molecular Biology Institute, UCLA School of Medicine, Los Angeles, CA, USA

    Q Liang & HR Herschman

  5. Department of Biomathematics, UCLA School of Medicine, Los Angeles, CA, USA

    MP Phelps & SS Gambhir

  6. UCLA-Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, CA, USA

    MP Phelps, SS Gambhir & HR Herschman

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Liang, Q., Satyamurthy, N., Barrio, J. et al. Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction. Gene Ther 8, 1490–1498 (2001). https://doi.org/10.1038/sj.gt.3301542

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