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  • The extent to which GPCR oligomerization is a regulated

    2023-04-14

    The extent to which GPCR oligomerization is a regulated process still remains unclear. Reported effects of ligands on both GPCR homo- and heteromers are highly variable and depend on GPCR subtypes and the specific ligand used. According to our data, chronic treatment of mice with both paroxetine or risperidone resulted in the significant increase of 5-HT1A–D2 receptor heteromer level in the PCX, unlike a single administration of paroxetine or risperidone, which did not induce any significant changes in the level of 5-HT1A–D2 receptor heteromers, when measured 30 min after i.p. drug administration. Chronic treatment with paroxetine or risperidone corresponds to the clinical practice, in which the drugs are administered over extended Hoechst 34580 of time. Although the onset of action for antipsychotic drugs is generally shorter than for antidepressant drugs (Agid et al., 2003, Machado-Vieira et al., 2010), full therapeutic benefits take several weeks to be achieved for both of these drug classes. What is worth emphasizing, the changes in PLA signals observed in situ after repeated paroxetine administration were similar to those observed after repeated low-dose risperidone treatment, although the pharmacological profiles of both drugs are different. Contrary to paroxetine, which has negligible affinity for serotonin 5-HT1A and dopamine D2 receptors, risperidone is a multireceptor antagonist, which among others has high affinity for D2 receptor (IC50 = 3.2 nM) and a moderate affinity for 5-HT1A receptor (IC50 = 420 nM) (Leysen et al., 1994). However, some clinical indications for use are similar for both studied drugs, i.e., in anxiety-related disorders. Paroxetine is a potent SSRI with established efficacy in the treatment of major depression, OCD, panic disorder, social anxiety disorder, posttraumatic stress disorder, and generalized anxiety disorder (Nemeroff, 1993, Ringold, 1994, Oehrberg et al., 1995, Zohar and Judge, 1996), while risperidone is a second-generation antipsychotic drug with proven efficacy as an anxiolytic agent, especially in low-dose treatment (Jacobsen, 1995, Saxena et al., 1996, Bartzokis et al., 2005, Brawman-Mintzer et al., 2005, Erzegovesi et al., 2005, Galynker et al., 2005, Gao et al., 2006; Padala et al., 2006, Simon et al., 2006). In our study the doses of drugs administered to mice were chosen based on the literature. Paroxetine in a dose 10 mg/kg exerts antidepressant, anticompulsive and anxiolytic activity in animals (Shinomiya et al., 2005, Nicolas et al., 2006, Mirza et al., 2007, Casarotto et al., 2010, Thoeringer et al., 2010, Huang et al., 2014), while risperidone in low-dose have anxiolytic effects in some behavioral tests (Sun et al., 2010, Rogóż and Skuza, 2011, Kamińska and Rogóż, 2016). As mentioned earlier, the PLA assay detects two proteins (here 5-HT1A and D2 receptors) located in very close physical proximity, but does not provide any clues on the total number of 5-HT1A and D2 receptors, when considered separately. In this regard, the density of 5-HT1A and D2 receptors was measured by ligand autoradiography in the same animals. Evaluating the receptor density with autoradiography performed at a single ligand concentration might not fully reflect the changes in receptors density, as there are some evidences for other types of GPCRs that forming the heteromers might lead to alterations in the protomer’s biochemical and/or pharmacological properties, i.e., ligand binding affinity or density, reviewed by Satake et al. (2013). Thus, we have complemented the ligand binding studies with measuring expression of mRNA encoding 5-HT1A and D2 receptors by in situ hybridization. The observed changes in 5-HT1A–D2 receptor heteromerization level after chronic paroxetine or risperidone treatment in the prefrontal cortex was not accompanied by any significant changes in the expression of 5-HT1A nor D2 receptors in that brain region. Therefore, it might indicate specific changes in the direct interaction between receptor protomers and facilitation of heteromers formation in the plasma membrane either by interaction with other co-localizing proteins and/or changes in membrane fluidity. The latter hypothesis has been currently validated by the observation that chronic antidepressant treatment may lose lipid rafts, and thereby facilitate G protein (or possibly also other proteins) accession to partner GPCR (Erb et al., 2016). Moreover, segregation of GPCRs in membrane microdomains (e.g., lipid rafts) was shown to regulate the formation of heteromeric complexes (Xiang et al., 2002).