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RGS2 Modulation of Dopamine D2 Receptor Internalization and Recycling in Neuroblastoma Cells
Author(s) -
Luessen Deborah Joyce,
Hinshaw Tyler,
Sun Haiguo,
Marrs Glen,
Chen Rong
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1190.15
Subject(s) - quinpirole , dopamine receptor d2 , internalization , regulator of g protein signaling , g protein coupled receptor , microbiology and biotechnology , agonist , chemistry , biology , g protein , receptor , dopamine , signal transduction , neuroscience , gtpase activating protein , biochemistry
Altered dopamine (DA) D2 receptor (D2R) expression and function are commonly implicated in the pathophysiology of drug addiction, Parkinson's disease and schizophrenia. Internalization and recycling of D2R play a key role in the regulation of D2R activity including desensitization and resensitization. Therefore, it is imperative to improve our understanding of the molecular mechanisms underlying D2R trafficking. Regulator of G‐protein signaling (RGS) proteins are known to modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. However, recent findings highlight non‐canonical roles of RGS proteins in modulation of various aspects of GPCR including trafficking. Methods In the present study, we utilized a neuroblastoma (N2A) cell model to assess the role of RGS2 (regulator of G‐protein signaling 2) in the modulation of D2R trafficking and signaling by knocking down RGS2 via siRNA. Immunocytochemistry and confocal microscopy were utilized to study both constitutive and receptor agonist (quinpirole) stimulated D2R internalization and recycling. The time courses of constitutive and quinpirole (1μM) stimulated D2R internalization and recycling were evaluated at numerous time points ranging from 0 to 90 minutes. Results Our data indicated that RGS2 knockdown increased constitutive D2R internalization and recycling when compared to the control cells treated with scrambled siRNA. When D2R was activated by the agonist quinpirole (1 μM), there was a reduction in surface D2R level in controls cells and an increase in surface D2R level in RGS2 knockdown cells. The reduced surface D2R level in control cells induced by quinpirole treatment was due to the accelerated D2R internalization which was both clathrin and dynamin‐dependent. Both dynasore, a dynamin inhibitor, and concanavalin A, a clathrin inhibitor, blocked quinpirole‐induced D2R internalization in control cells. In contrast, RGS2 knockdown significantly attenuated quinpirole‐induced D2R internalization. Furthermore, the D2R recycling rate following quinpirole treatment in RGS2 knockdown cells was 2.5 times greater than that in the control cells. The effect of quinpirole on D2R surface trafficking was G protein dependent because pertussis toxin treatment abolished induced D2R trafficking. We also examined whether the downstream signaling molecule pERK mediates D2R internalization and recycling in the RGS2 knockdown cells. These results add a novel insight to the role of RGS2 in modulation of D2R internalization and recycling and may highlight the novel mechanistic actions of RGS2 in mediation of D2R trafficking. Support or Funding Information This work is supported by the National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health T32AA007565 training grant, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.