Effects of the Dopamine D2 Allosteric Modulator, PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

Abstract

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.

Calcium-Sensing Receptor Internalization Isβ-Arrestin–Dependent and Modulated by Allosteric Ligands

“…In the present study, we show that CaSR internalization is negatively and positively modulated by NPS 2143 and NPS R-568, respectively. These results are in agreement with previous findings on other receptors reporting allosteric modulation of ligand-mediated internalization for the class A M1 and M4 muscarinic acetylcholine receptors (Leach et al, 2010;Yeatman et al, 2014), D2 dopamine receptor (Basu et al, 2013), cannabinoid receptor type 1 (Ahn et al, 2012;Laprairie et al, 2015), and the class C metabotropic glutamate receptor 7 (Pelkey et al, 2007). Furthermore, NPS R-568 triggered a small but statistically significant increase in constitutive CaSR internalization.…”

Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA

“…Previous studies from our lab have demonstrated the downstream effect of PAOPA on kinases, such as extracellular signal-regulated kinase 1/2 (ERK 1/2) [1]. Therefore, expression of synapsin, as observed in our studies could also be a downstream modulatory result of ERK1/2, as has been shown to occur in previous studies [22,33].…”

“…Stimulation of D1 receptor activity by agonists, or attenuation of D2 activity by antagonists, results in increased synapsin II levels [11,41]. The use of PAOPA, has previously been shown to promote internalization of the D2R, thereby decreasing overall availability of this receptor, similar to an antagonist, but with a more fine-tuned and subtle technique [1]. Reduced post-synaptic D2 activity caused by PAOPA-induced receptor internalization within the striatum, NAc and mPFC, is hypothesized to lead to decreased inhibition of cyclic adenosine monophosphate (cAMP), causing increased protein kinase A (PKA) stimulation, which eventually results in Ap-2␣ transcription factor promoted enhancement of synapsin II transcription [41].…”

“…Characterization of PAOPA's pharmacological properties has revealed that it modulates agonist binding to dopamine D2 and D4 receptors in a biphasic manner [48]. PAOPA has also shown to promote D2R internalization, and upregulate proteins involved in this process, revealing initial processes linked to its potential mechanism of therapeutic action [1]. However, the effect of PAOPA on the expression of the crucial presynaptic secretory proteins mentioned above is currently unknown.…”

Dopamine receptors – IUPHAR Review 13

“…Thus, a specific allosteric modulator of D 2 receptors, the peptidomimetic 3(R)‐[(2(S)‐pyrrolidinylcarbonyl) amino]‐2‐oxo‐1‐pyrrolidineacetamide (PAOPA), has been recently characterized as binding to a site on the D 2 receptor that is distinct from the endogenous ligand binding site (Tan et al ., ). PAOPA can influence several signalling and cellular events characteristic of D 2 receptor activation (Basu et al ., ), and in preclinical models, can attenuate schizophrenia‐like behavioural manifestations without causing significant motor abnormalities common with many current antipsychotics (Tan et al ., ).…”