Effects of the dopamine D2 allosteric modulator, PAOPA, on the expression of GRK2, arrestin-3, ERK1/2, and on receptor internalization.
The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA's effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA's development into a novel drug for the improved treatment of schizophrenia.
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada.
Dipannita
D
Basu
Yuxin
Y
Tian
Jayant
J
Bhandari
Jian Ru
JR
Jiang
Patricia
P
Hui
Rodney L
RL
Johnson
Ram K
RK
Mishra
D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D019390:Arrestins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000378:metabolism*
D019390:Arrestins; D011760:Pyrrolidinones; D017448:Receptors, Dopamine D2; C082333:arrestin3; C067487:3-(N-prolylamine)-2-oxo-1-pyrrolidineacetamide; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3