Investigating the role of spinophilin in mediating Dopamine D2 Receptor Activity

Abstract ID 94088 Poster Board 321 Striatal dysfunction is linked to several conditions, including Parkinson's Disease, attention-deficit hyperactivity disorder (ADHD), and substance use disorders. Psychostimulants such as methamphetamine or amphetamine are employed in the treatment of disorders associated with the striatum, such as ADHD. These medications induce long lasting alterations in the striatum by modulating dopamine transmission, but also have a high abuse potneital. Dopamine 2 receptors (D2Rs) are G-protein coupled receptors, that respond to excessive dopamine release induced by psychostimulants. However, the neuroadaptive changes in D2R function caused by chronic receptor activation associated with ADHD or PD treatments, are unclear. Previous studies have found that spinophilin, the major synaptic protein phosphatase 1 (PP1) targeting protein, directly binds the third cytoplasmic loop of the D2R. Additionally, the loss of spinophilin causes deficits in rotarod learning and have suppressed effects of amphetamine induced locomotor sensitization. However, how spinophilin modulates D2R phosphorylation and function is not known. Our preliminary data show that loss of spinophilin enhances the tolerance to the locomotor suppressive effects of the D2R agonist quinpirole. In addition, excessive motor output in a mouse model for repetitive grooming demonstrated an increase in the interaction between spinophilin and the D2R. However, how spinophilin modulates D2R function is unclear.To investigate our hypothesis we need to generate a novel approach to isolate D2R that is transiently-associated with spinophilin. To achieve this, we have generated and validated a spinophilin-UltraID fusion protein that we have transfected into HEK293 cells. Our ongoing studies in HEK293 cells will utilize these constructs to delineate how spinophilin modulates D2R phosphorylation and protein interactions as well as how D2R agonists modulate D2R function in the absence or presence of spinophilin. This characterization will lay the foundation for our ongoing animal studies to generate a transgenic mouse that will permit Cre-dependent expression of an endogenous spinophilin-Ultra ID protein. In addition, these tools will complement our currently used global and cell type-specific spinophilin knockout mice. Overall, this project advances our knowledge of spinophilin-dependent regulation of the D2R, a drug target for various diseases, such as PD and ADHD.