Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Abraham Kovoor

Abstract

Clozapine is an antipsychotic drug (APD) with a uniquely efficacious therapeutic profile that is preeminent for treatment resistant schizophrenia. A common property of all clinically available APDs is that they bind D2-like dopamine receptors at therapeutic concentrations. However, unique molecular actions for clozapine that are different from all other APDs have not been established. Here we show that treatment with all APDs upregulates cell-surface expression of D2-dopamine receptors (D2R), relative to that produced by clozapine. Multiple experiments, including a pulse-chase assay for surface D2R indicated that the APD-mediated upregulation of D2R is due to enhanced insertion of D2R into the plasma membrane and not differences in receptor occupancy or protein synthesis. The potency of amisulpride, an APD with poor membrane permeability, for upregulating surface D2R was increased by coexpressing OCT1, an amisulpride transporter, suggesting an intracellular site of action for APDs. Competition experiments and experiments with a non-APD-binding D2R mutant indicated that APD upregulation of surface D2R is a consequence of direct receptor binding. Enhancement by APDs of surface D2R, relative to that produced by clozapine, was magnified in both a glycosylation-deficient and a misfolding D2R mutant. Moreover, every APD, except clozapine, enhanced the detergent solubility of intracellular D2R. These and other data suggest that APDs act intracellularly as chemical chaperones to stabilize a conformation of D2R with enhanced detergent solubility and transport to the cell surface. These results display a unique molecular event of clozapine at D2R, as it possesses the lowest efficacy with respect to these actions of APDs. Results displaying unique binding of clozapine to D2Rharbor intriguing implications for APD discovery and development, and provokes novel speculative thought and questions on the nature of ligand-receptor interaction.

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