Zein, Josephine2022-08-092022-08-092022-08-09http://hdl.handle.net/10393/43895http://dx.doi.org/10.20381/ruor-28108Compromised D1R-class signalling is implicated in several neurodegenerative disorders such as in the case of L-DOPA-induced dyskinesia (LID) seen in individuals suffering from Parkinson’s disease (PD). Therefore, extensive research is required to be able to therapeutically target the altered mechanisms in D1R-class responsiveness which develop in these psychomotor disorders. One main component that is yet to be fully delineated is the role of proteins that interact with the intracellular loops of the D1R-class. Thus, to gain further insight into the protein-protein interaction networks implicated in the regulation of the D1R-class, our laboratory performed yeast two-hybrid screening to characterize the interactions between PLZF and the cytoplasmic tail of the D1R, as well as PLZF and the third intracellular loop of the D5R. Furthermore, our lab identified endogenous PLZF-D5R complex formation within the rat hippocampus and striatum. Interestingly, PLZF is described as a multifunctional transcription regulator and has been characterized as a cytosolic and nuclear protein expressed in developing and mature brains. PLZF has also been shown to interact with the agonist-stimulated GPCR AT2R, internalize with AT2R, shuttle to the nucleus upon prolonged AngII exposure and bind to the constitutively activated Gαo subunit. Based on these findings, I hypothesized that PLZF regulates D1R-class function through complex formation, signaling and trafficking mechanisms. Herein, I demonstrate using co-transfected HEK293 cells that upon agonist exposure, there is an increase in the association between PLZF and the D1R-class. The PLZF-D1R and PLZF-D5R interactions do not alter the ligand binding affinities of the receptors. Interestingly, the co-expression of β-arrestin 2 with PLZF and either the D1R or D5R results in decreased recruitment of β-arrestin 2 to the receptors. Furthermore, the PLZF-D1R interaction results in increased cAMP and ERK activation, while PLZF-D5R interaction results in decreased cAMP and ERK activation. PLZF also increases the cellular surface and total expression of the D1R but does not have any effect on the D5R surface and total expression. The distinct contrasting effect of PLZF interaction with the D1R-class is also observed in the disrupted internalization of the D1R vs. the facilitated internalization of the D5R. Moreover, β-arrestin 2 co-expression with PLZF impedes the extent of internalization for both the D1R and D5R. Finally, PLZF distinctly regulates D1R-class recycling by possibly accelerating D1R recovery and delaying D5R recovery. These novel findings may provide a better understanding of the molecular mechanisms involved in the etiology of neuropsychiatric diseases and potentially facilitate the development of treatments for dopamine-related disorders.enThesis