The effect of clozapine on mRNA expression for genes encoding G protein-coupled receptors and the protein components of clathrin-mediated endocytosis

abstract: ...Objectives Clathrin-mediated endocytosis (CME) is an intracellular trafficking mechanism for packaging cargo, including G protein-coupled receptors (GPCRs), into clathrin-coated vesicles (CCVs). The antipsychotic chlorpromazine inhibits CCV assembly of adaptor protein AP2 whereas clozapine increases serotonin2A receptor internalization. We hypothesized that clozapine alters the expression of CME genes modulating vesicle turnover and GPCR internalization. Materials and methods SH-SY5Y human neuroblastoma cells were incubated with clozapine (120 mol/l) for 2472 h. GPCR and CME-related gene mRNA expression was measured using RT-PCR. We quantified changes in the same genes using expression data from a microarray study of mice brains after 12 weeks of treatment with 12 mg/kg/day clozapine. Results The expression of genes encoding adaptor and clathrin assembly proteins, AP2A2 , AP2B1 , AP180 , CLINT1 , HIP1 , ITSN2 , and PICALM , increased relative to the control in SH-SY5Y cells incubated with 510 mol/l clozapine for 2472 h. The microarray study showed significantly altered expression of the above CME-related genes, with a marked 641-fold and 17-fold increase in AP180 and the serotonin1A GPCR, respectively. The expression of three serotonergic receptor and lysophosphatidic acid receptor 2 ( EDG4 ) GPCR genes was upregulated in SH-SY5Y cells incubated with 5 mol/l clozapine for 24 h. EDG4 expression was also increased with 1020 mol/l clozapine treatment at 4872 h. Clozapine significantly decreased the expression of -arrestin, involved in GPCR desensitization, both in vitro and vivo . Conclusion The changes we report in CME and GPCR mRNAs implicate CCV-mediated internalization of GPCRs and the serotonergic system in clozapines mechanism of action, which may be useful in the design of more effective and less toxic antipsychotic therapies.

... measured using RT-PCR. We quantified changes in the same genes using expression data from a microarray study of mice brains after 12 weeks of treatment with 12 mg/kg/day clozapine. Results The expression of genes encoding adaptor and clathrin assembly proteins, AP2A2 , AP2B1 , AP180 , CLINT1 , HIP1 , ITSN2 , and PICALM , increased relative to the control in SH-SY5Y cells incubated with 510 mol/l clozapine for 2472 h. The microarray study showed significantly altered expression of the above CME-related

...) using cDNA replicates (i.e. not pooled). In addition, qPCR assays were designed for seven CME-related genes including adaptor-related protein complex 2, 2 subunit ( AP2A2 ), adaptor-related protein complex 2, 1 subunit ( AP2B1 ), AP180 , CLINT1 , disabled homolog 1 and 2 ( DAB1 , DAB2 ), Huntingtin interacting protein 1 ( HIP1 ), intersectin 2 ( ITSN2 ), and phosphatidylinositol-binding clathrin assembly lymphoid myeloid protein ( PICALM ). RT-qPCR GPCR, CME, and reference gene expression assays

... : KruskalWallis, 2 =6.333, d.f. =5, P =0.275; AP180 : one-way ANOVA, F =1.141, P =0.360; HIP1 : one-way ANOVA, F =0.585, P =0.712; ITSN2 : one-way ANOVA, F =1.745, P =0.149; PICALM : one-way ANOVA, F =0.451, P =0.809). There were no significant differences between the treatment group means for AP2A2 and CLINT1 genes at any time point (one-way ANOVA, P >0.05). Table 2 SH-SY5Y neuroblastoma gene expression following treatment with clozapine (120 mol/l) for 24, 48, and 72 h GPCR gene expression screening

...-1 angiotensin II receptor-associated protein; AGTR1 , angiotensin II type-1B receptor; AGTR2 , angiotensin II type-2 receptor; AP180 , clathrin coat assembly protein 180, also known as synaptosomal-associated protein, SNAP91; AP2A2 , adaptor-related protein complex 2, 2 subunit, AP2B1 , adaptor-related protein complex 2, 1 subunit; ARRB2 , arrestin -2; CCR5 , chemokine (CC motif) receptor 5; CDKN1B , cyclin-dependent kinase inhibitor 1B; CLINT1 , clathrin interactor 1; DRD1 , dopamine D1

... 12-week chronic treatment with clozapine or vehicle can be downloaded from http://www.ucl.ac.uk/~rejuhxg/ClozHalStudy/ . Clozapine significantly altered the expression of 1330 genes relative to control. Clozapine altered the expression of nine GPCR and CME genes in the mouse brain with increases in AP2A2 by 3.8-fold (MANOVA, F =46.527, d.f. =4, N =76, P =6.1610 8 ), AP2B1 by 2.7-fold (MANOVA, F =98.520, d.f. =2, N =40, P =4.4610 8 ), AP180 by 641.0-fold (MannWhitney U -test, Z =3.782, N =20, P

..., d.f. =3, N =60, P =1.8510 14 ) (Fig. 3 shows the logged mRNA fold change). Fig. 3 Log 10 transformation of fold change in clathrin-coated vesicles and G protein-coupled receptor gene expression in mouse brain following treatment with 12 mg/kg/day clozapine for 12 weeks: data from a microarray study. AP180 , clathrin coat assembly protein 180, also known as synaptosomal-associated protein, SNAP91; AP2A2 , adaptor-related protein complex 2, 2 subunit; AP2B1 , adaptor-related protein complex 2, 1

... cell surface expression of multiple GPCRs and their trafficking through different clathrin adaptors. We found significant increases in the gene expression of AP2A2 after 24 h along with CLINT1 and clathrin assembly proteins AP180 and PICALM after 72 h of clozapine treatment in SH-SY5Y cells compared with the control. Clozapine increased AP2B1 , AP180 , HIP1 , ITSN2 , and PICALM gene expression in a dose-dependent manner at both 48 and 72 h. These increases in mRNA levels are in keeping with the

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Gene: AP2A2 - adaptor-related protein complex 2, alpha 2 subunit - (NCBI)

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