Student Theses and Dissertations

Date of Award

2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory

Abstract

The human dopamine receptor 4 (hD4R) is a seven-transmembrane helical G protein-coupled receptor (GPCR) found in neural synaptic membranes. The neurotransmitter dopamine binds to and activates the hD4R, which is involved in central nervous system pathways that modulate cognition and circadian rhythms. The hD4R is the primary dopaminergic receptor for the atypical anti-psychotic drug clozapine, which is used to treat schizophrenia and other cognitive disorders. The hD4R gene is unique among the superfamily of GPCR-encoding genes because within the human population, it contains a variable number of tandem repeat (VNTR) exon polymorphism. Because of the VNTRs, the length of the primary structure of one of the intracellular loops of the hD4R can vary dramatically among individuals. Attempts have been made to correlate different VNTR structures with different behavioral traits – for example, a specific variant of hD4R is robustly correlated with attention deficit hyperactivity disorder. Like other GPCRs, hD4R functions at the plasma membrane by binding an extracellular ligand, in this case dopamine, to regulate an intracellular signaling cascade. The density of hD4R at the plasma membrane and its distribution within the neuron/synapse dictate the cellular response to dopamine. Despite the importance of hD4R in neuronal signaling, the molecular mechanisms regulating its cellular expression and degradation are unclear. Isopeptide ubiquitination of lysine residues on the cytoplasmic surface of various GPCRs regulates receptor abundance at the membrane by promoting protein degradation. I have studied the role of the ubiquitin-proteasome system in the cellular degradation of hD4R, and show here that hD4R protein levels are regulated through both a canonical and a non-canonical ubiquitination pathway. Site-directed mutagenesis of lysine residues, as well as mutagenesis of the atypical ubiquitin acceptors serine and threonine, led to an additive increase in mutant hD4R protein abundance in a cellular expression model. Chemical inhibition of the proteasome increased levels of the wild-type hD4R, but not the lysine, serine, and threonine null mutant. Both isopeptide ubiquitination of lysine and ester bond ubiquitination of serine and threonine were detected on hD4R in a model protein expression system using immunoprecipitation techniques. A proximity ligation assay was used to quantify isopeptide and ester bond ubiquitination in this protein expression system and to detect ubiquitination of hD4R in mouse primary cortical neurons. Together, these data support the hypothesis that hD4R is proteasomally degraded after isopeptide ubiquitination of lysine residues and ester ubiquitination of serine and threonine residues. The ubiquitination and subsequent degradation of hD4R represents a mechanism for cellular control over hD4R protein levels. While the low abundance of hD4R protein produced in heterologous expression systems has previously been limiting for biochemical and structural biology techniques, the degradation-resistant hD4R mutants presented here overcomes this limitation and may facilitate future research, including the identification of dopamine receptor interacting proteins (DRIPs). hD4R joins a small number of proteins that are known to be modified by ubiquitination via ester bonds. This work also describes novel techniques to confirm and quantify ester-bond ubiquitination for a given membrane protein within a cell.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for The degree of Doctor of Philosophy

Available for download on Wednesday, November 13, 2019

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