Author ORCID Identifier

https://orcid.org/0000-0002-0648-1824

Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Neuroscience

First Advisor

Javier Gonzalez-Maeso, PhD

Second Advisor

Clive M. Baumgarten, PhD

Third Advisor

Dr. Kurt F. Hauser, PhD

Fourth Advisor

Gretchen N. Neigh-McCandless, PhD

Fifth Advisor

Ian Scott Ramsey, PhD

Abstract

Class A serotonin (5-hydroxytryptamine) 2A (5-HT2AR) and class C metabotropic glutamate 2 receptors (mGluR2) are seven transmembrane receptors (7TMRs or G protein-coupled receptors – GPCRs) involved in multiple neuropsychiatric disorders including schizophrenia. Previous findings from our laboratory reported that 5-HT2AR and mGluR2 are dysregulated in the prefrontal cortex of patients suffering from this psychiatric condition, although 5-HT2AR’s expression was recovered in antipsychotic-medicated patients. Genome-wide association studies on schizophrenia reported that endosomal trafficking that regulates cell surface abundance of another 7TMR implicated in this disease (dopamine D2 receptor) can be altered. Ligand-activated receptors, including the 7TMR superfamily, are dynamic entities whose signaling depends on trafficking, and trafficking on signaling. Impairments in the spatial and temporal regulation of 7TMRs result in homeostasis perturbations and later in diseases. Like other cell surface proteins expressed in mammals, such as enzyme-associated receptors and ligand-gated ion channels, 7TMRs form homo- or hetero-oligomers, conferring novel and unique functional properties to the complex. Our previous findings suggested that 5-HT2AR and mGluR2 are assembled in a functional GPCR heteromeric complex in both heterologous expression systems and in mouse and human brain frontal cortex. However, evidence that GPCR heteromerization can directly affect receptor subcellular organization and localization is missing, especially across classes. Here, we showed in mammalian HEK293 cells that 5-HT2AR affected the localization and trafficking of mGluR2 through a mechanism that required their physical assembly. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments. Intriguingly, the plasma membrane-localized mGluR2 was redistributed in internal compartments when coexpressed with 5-HT2AR. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5- or Rab7-positive endosomes in cells expressing the receptors alone, or together. In addition, overnight blockade of 5-HT2AR with antipsychotic clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved 5-HT2AR-mGluR2 heteromerization. Using TAT-tagged interfering peptides and a chimeric construct unable to heteromerize with 5-HT2AR, we showed that heteromerization was necessary for the 5-HT2AR-mediated co-trafficking of mGluR2. Furthermore, using bimolecular fluorescence complementation (BiFC), we tracked the maturation of the 5-HT2AR-mGluR2 complex in the endoplasmic reticulum and the Golgi apparatus. In accordance with our findings in HEK293 cells, mGluR2/Rab5 colocalization was diminished in neuronal cortical primary culture of 5-HT2AR-/- mice as compared to controls. Additionally, mGluR2 was mostly localized near the plasmalemma in brain sections of 5-HT2AR-/- mice frontal cortex glutamatergic excitatory postsynaptic compartments. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

Comments

Link to published article: https://www.science.org/doi/10.1126/scisignal.aaw3122?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

12-15-2021

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