The organic cation Transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus

Abstract
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Article and author information

Abstract

Dopamine is a key catecholamine in the brain and the kidney, where it is involved in a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation and renal function. As a membrane impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein couple receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on Organic Cation Transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in murine striatal medium spiny neurons (MSN) and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gas coupling, using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data provide further evidence for the role of transporters in regulating subcellular GPCR activity.

Data availability

Source Data has been provided for Figures 1c, 1d, 2b, 2c, 2f, 2g, 3c, 4d, 4f, 5c and 6c as well as Figures 1-Figure Supplement 1b-d, Figure 1-Figure Supplement 3b, Figure 1-Figure Supplement 4c, Figure 2-Figure Supplement 1a-b, Figure 3-Figure Supplement 1a. We have also included information for primers, shRNAs and plasmid maps. Antibodies, cells and reagents are also provided in the source data.

Article and author information

Author details

Natasha M Puri

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Giovanna R Romano

Biochemistry Department, Weill Cornell Medicine, New York, United States

Competing interests
The authors declare that no competing interests exist.
Ting-Yu Lin

Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Quynh N Mai

Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6199-2096
Roshanak Irannejad

Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

For correspondence
roshanak.irannejad@ucsf.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8702-2285

Funding

NIH Office of the Director (GM133521)

Roshanak Irannejad

The American Heart Association (Graduate student fellowship)

Natasha M Puri

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal tissues from mice were isolated as recommended and approved by the institutional animal care and use committee (IACUC protocol #AN184251).

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.