PLZF as a Novel Regulator of Dopamine D1 Receptor Signaling

Abstract

G protein-coupled receptors (GPCRs) represent the largest and most diverse family of transmembrane receptors, mediating a wide range of physiological processes through the activation of intracellular signaling cascades. Among these, dopamine receptors (DARs) play a central role in regulating critical neurological functions such as movement, cognition, and reward. The D1-class dopamine receptors (D1R and D5R), particularly the D1R, have been implicated in several neurological disorders such as Parkinson’s Disease (PD) and Levodopa-Induced Dyskinesia (LID). Therefore, more research is needed to better understand novel methods to be able to treat these dysfunctions. A major topic in D1R signaling is the role of proteins that interact with the receptor intracellular domains. The Promyelocytic Leukemia Zinc Finger (PLZF) protein was previously identified through yeast two-hybrid screening in the Tiberi lab as a novel interacting partner of the D1-class receptors. Unpublished studies in our lab found that PLZF, a multifunctional nuclear protein and transcriptional regulator with major roles in brain development, exerts subtype-specific effects on D1-class signaling and trafficking. Previous studies have also shown that PLZF forms a complex with both the agonist-stimulated GPCR AT2R and the constitutively activated Gαo subunit. However, its mechanistic role in D1R signaling and the desensitization process remains poorly understood. In this thesis, I investigate the phosphorylation of the proximal cytoplasmic tail (CT) residues of the D1R, focusing on the contribution of the more distal CT domains in addition to complex formation with PLZF. Using HEK293 cells transfected with Flag-tagged wild-type or truncated D1R constructs (fVal388-STOP, fSer417-STOP, fSer431-STOP), receptor phosphorylation was assessed at specific proximal residues (Thr354 and Ser372/Ser373) through immunoprecipitation studies following dopamine stimulation. The results demonstrate that truncation of the distal domains of the D1R CT sequentially impairs phosphorylation of upstream residues, suggesting a hierarchical phosphorylation pattern. Co-expression of HA-tagged PLZF with D1R significantly decreases phosphorylation at these proximal sites, and this effect was modulated in truncated receptors, indicating that PLZF-mediated regulation occurs via specific distal domains of the CT. Furthermore, GST pulldown assays revealed that PLZF interacts with both the IL3 and CT domains of D1R, with PLZF exhibiting a stronger affinity for the CT. Additionally, co-immunoprecipitation studies show that PLZF displays dynamic recruitment to the IL3 and CT, with truncation of either IL3 domain modulating the degree of binding under basal and dopamine-stimulated states, as well as retainment of recruitment with complete removal of the CT. Collectively, this work identifies PLZF as a key regulator of D1R signaling and provides new insight into the molecular mechanisms controlling dopaminergic receptor responsiveness. These novel findings may provide a better understanding of the molecular underpinnings involved in disorders compromising the dopaminergic system and potentially inform future therapeutic strategies for dopaminergic dysfunction in neurological disorders.