Additionally regulation of GPCR heteromerization by

Additionally, regulation of GPCR heteromerization by specific ligands may depend not only on the affinity of ligands for receptors, but also on presence of different interacting proteins co-localizing with the receptors in the specific cell, which may induce some conformational changes and contribute significantly to the formation of GPCR homo- and heteromers. It was shown that antipsychotic treatment may induce specific changes in expression of dopamine D2 receptor interacting proteins in the Deforolimus sale (Kabbani and Levenson, 2006). Another factor that is reported to modulate the oligomeric structure of some receptors is the activation state of the G proteins to which they are coupled. For example, inactivation of receptor–G protein interactions induces the dissociation of SST2 homomers similarly to agonist stimulation, while it has no effect on ligand-induced homodimerization of the SST5 receptor (Grant et al., 2004). The fact, that paroxetine has a negligible affinity for 5-HT1A and D2 receptors (Thomas et al., 1987), but is able to influence 5-HT1A–D2 receptor heteromerization in vivo after chronic paroxetine treatment, may suggest that similar co-localizing interacting proteins may have some role in the observed changes in 5-HT1A–D2 heteromerization level after paroxetine treatment. Recently, it has been shown that paroxetine, besides being a potent blocker of serotonin transporter, is a direct inhibitor of G-protein-coupled kinase 2 (GRK2) (Thal et al., 2012, Schumacher et al., 2015), what has its significant molecular consequences, such as inhibiting β-adrenergic receptor phosphorylation, decreasing β-arrestin recruitment and down-regulating internalization of adrenergic receptors in response to catecholamine stimulation (Guo et al., 2017), what all leads to diminished β-adrenergic desensitization. Complementary to in situ PLA, for studies of 5-HT1A–D2 heteromerization we used homogenous time-resolved FRET (HTRF) technology applied to in vitro cell system and human 5-HT1A and D2 receptors. Similarly to other antipsychotics which were tested earlier (Łukasiewicz et al., 2016), risperidone treatment resulted in the increase of the level of 5-HT1A–D2 heteromers. Likewise, paroxetine treatment also resulted in the increase of 5-HT1A–D2 heteromerization. The data obtained in vitro further extended the significance of in situ PLA results, as both techniques (i.e., in situ PLA and in vitro HTRF) confirm the influence of paroxetine and risperidone on 5-HT1A–D2 heteromerization level and extended the significance of the obtained effects to human 5-HT1A and D2 receptors. As revealed by previous in vitro study, the increase in level of 5-HT1A–D2 heteromerization after antipsychotic drugs treatment has its functional consequences, such as increased ERK1/2 activity (Łukasiewicz et al., 2016), which might regulate synaptic plasticity, brain development and repair as well as memory formation (Peng et al., 2010, Sun and Nan, 2017).
Conclusion As revealed by our immunofluorescent staining and in situ PLA, the co-localization and the potential interaction of 5-HT1A and D2 receptors occurred in the mouse prefrontal cortex. Counterstaining with GFAP and NeuN after PLA confirmed the astrocytal and neuronal localization of 5-HT1A–D2 receptor heteromers. Although paroxetine and risperidone possess different pharmacological profiles, after chronic treatment they induced similar changes in 5-HT1A–D2 receptor heteromerization level in situ in the prefrontal cortex and in vitro in heterologous cell system. The obtained results implicate the common mechanism in paroxetine and risperidone mode of action.
Conflict of interest
Acknowledgments The study was financially supported by National Science Centre Grant No. PRELUDIUM UMO-2012/05/N/NZ7/00664, Poland and Statutory Activity of Institute of Pharmacology Polish Academy of Sciences, Poland. Publication charge was supported by KNOW (Krajowy Naukowy Osrodek Wiodacy) funds MNiSW-DS-6002-4693-26/WA/12, Poland. Żurawek D. is a holder of START 2017 scholarship supported by the Foundation for Polish Science (FNP), Poland. All of Institutions had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.