An important observation in our study is the lack of

An important observation in our study is the lack of effect of paroxetine pretreatment alone on β2AR membrane expression, which suggests that paroxetine, and pharmacologic inhibition of GRK2 in general, does not directly induce changes in receptor dynamics. Rather, the decreased ligand-induced β-arrestin recruitment and receptor internalization following stimulation would be attributable to the lack of GRK2-mediated receptor phosphorylation. This fits with the known requirement for GRK2 to be recruited to the receptor after ligand stimulation [21], thus its inhibition prior to β2AR stimulation would not be expected to alter membrane-localized desensitization signaling dynamics. Although there are no reports directly comparing the impact of paroxetine on receptor 99026 amongst distinct GPCRs, in terms of relative efficacy paroxetine inhibited ligand-induced β2AR phosphorylation in our study with an IC50 of ~6μM, compared to an IC50 of ~30μM for the TRHR previously reported [7], possibly suggesting more reliance upon GRK2 for β2AR versus TRHR phosphorylation. Of note, a recent study showed that β2AR phosphorylation induced by hypoxic stress was also decreased by the GRK2 inhibitor 182,200 [22], however the concentration of inhibitor 182,200 required to partially block the response was 125μM, as compared to the 10–30μM range of paroxetine sufficient to almost completely abrogate β2AR phosphorylation in our study. More in line with our study, Lowe et al. demonstrated a similar impact of 30μM Takeda Cmpd 101 on ligand-induced μ-opioid receptor phosphorylation, βarr2 recruitment and receptor internalization [23]. Thus, paroxetine appears to have similar, or better, potency for GRK2 inhibition compared with other commercially-available small molecule antagonists.
Conclusions In summary, our results confirm that paroxetine blocks proximal signaling effects at βARs including receptor phosphorylation, βarr recruitment and internalization, providing a mechanistic link between previous structural/biochemical studies identifying paroxetine as a GRK2 inhibitor [7], [24], [25] and subsequent functional/pathophysiologic studies showcasing its effectiveness at reversing cardiac dysfunction [8], [18]. Thus, paroxetine inhibits βAR desensitization mechanisms consistent with GRK2 inhibition and provides a useful pharmacological tool for studying these facets of receptor regulation. This is highly relevant as efforts continue to develop next generation small molecule compounds with higher potency and efficacy toward GRK2 inhibition based on the structural properties of paroxetine [26].
Sources of funding
Disclosures
Introduction G-protein coupled receptors (GPCRs) constitute a superfamily of cell surface receptors characterized by seven tandemly-arranged transmembrane regions. Upon stimulation with agonists, activated GPCRs initiate intracellular signaling-mediated events, such as desensitization, endocytosis, and intracellular trafficking. The phosphorylation of GPCRs plays an important role in these events [1], [2]. Putative GPCR phosphorylation sites predicted based on sequence motifs in these receptors have been validated using site-directed mutational analysis in cell lines [3]. In vitro reconstitution experiments provide valuable information on phosphorylation sites; however, they cannot necessarily identify residues phosphorylated in living organs [4]. GPCRs are phosphorylated at multiple sites to regulate signaling in distinct tissues [5], [6]. Therefore, there might be significant differences in phosphorylation events between in vitro and in vivo conditions. Although direct analysis of in vivo natural phosphorylation states is particularly important, it is exceedingly difficult due to the low level of phosphorylated proteins in physiological conditions [7], [8]. In addition, analysis in tissues is very challenging owing to the complexity and wide dynamic range of phosphorylated peptides.