Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening
Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal contr...
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| Format: | Article |
| Language: | English |
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MDPI AG
2019-11-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/8/11/1468 |
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| author | Trevor B. Doyle Brian S. Muntean Karin F. Ejendal Michael P. Hayes Monica Soto-Velasquez Kirill A. Martemyanov Carmen W. Dessauer Chang-Deng Hu Val J. Watts |
| author_facet | Trevor B. Doyle Brian S. Muntean Karin F. Ejendal Michael P. Hayes Monica Soto-Velasquez Kirill A. Martemyanov Carmen W. Dessauer Chang-Deng Hu Val J. Watts |
| author_sort | Trevor B. Doyle |
| collection | DOAJ |
| description | Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein—NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D<sub>1</sub>- and D<sub>2</sub>-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms. |
| first_indexed | 2024-03-12T18:28:46Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T18:28:46Z |
| publishDate | 2019-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-2816566697a9482f908b17dccf107e2f2023-08-02T08:23:40ZengMDPI AGCells2073-44092019-11-01811146810.3390/cells8111468cells8111468Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation ScreeningTrevor B. Doyle0Brian S. Muntean1Karin F. Ejendal2Michael P. Hayes3Monica Soto-Velasquez4Kirill A. Martemyanov5Carmen W. Dessauer6Chang-Deng Hu7Val J. Watts8Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USADepartment of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USAMedicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USAMedicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USAMedicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USADepartment of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USADepartment of Integrative Biology & Pharmacology, McGovern Medical School, University of Texas Health Science Center, Houston, TX 77030, USAMedicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USAMedicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN 47906, USAAdenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein—NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D<sub>1</sub>- and D<sub>2</sub>-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms.https://www.mdpi.com/2073-4409/8/11/1468adenylyl cyclaseac5bifccampdopaminepp2astriatumnapa |
| spellingShingle | Trevor B. Doyle Brian S. Muntean Karin F. Ejendal Michael P. Hayes Monica Soto-Velasquez Kirill A. Martemyanov Carmen W. Dessauer Chang-Deng Hu Val J. Watts Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening Cells adenylyl cyclase ac5 bifc camp dopamine pp2a striatum napa |
| title | Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening |
| title_full | Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening |
| title_fullStr | Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening |
| title_full_unstemmed | Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening |
| title_short | Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D<sub>1</sub> and D<sub>2</sub> Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening |
| title_sort | identification of novel adenylyl cyclase 5 ac5 signaling networks in d sub 1 sub and d sub 2 sub medium spiny neurons using bimolecular fluorescence complementation screening |
| topic | adenylyl cyclase ac5 bifc camp dopamine pp2a striatum napa |
| url | https://www.mdpi.com/2073-4409/8/11/1468 |
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