Investigating the role of palmitoylation in the function of the dopamine D2 receptor.
Open Access
- Author:
- Ebersole, Brittany Anne
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 30, 2015
- Committee Members:
- Robert G Levenson, Dissertation Advisor/Co-Advisor
Faoud T Ishmael, Committee Member
Patricia Sue Grigson, Committee Member
Thomas E Spratt, Committee Member - Keywords:
- palmitoylation
dopamine D2 receptor
GPCR
click chemistry
immunoprecipitation
yeast two-hybrid
palmitoyl acyltransferases - Abstract:
- The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is crucial for regulation of processes such as mood, reward, and motor control. Abnormalities in D2R expression and/or neurotransmission are associated with a variety of human disorders, including schizophrenia, Parkinson’s disease, bipolar disorder, depression, and drug abuse. Palmitoylation, the thioester attachment of palmitate to cysteine residues, has become a topic of interest for GPCRs as it has been shown to modulate signaling, trafficking, and stability of these receptors. In this dissertation, an optimized version of bioorthogonal click chemistry (BCC) was developed with the ultimate goal of studying D2R palmitoylation. With this technique, proteins are labeled with the chemical probe 15-hexadecynoic acid (15-HDYA) at sites of palmitoylation. Proteins of interest are then isolated by immunoprecipitation with magnetic beads and analyzed for 15-HDYA incorporation. The utility of this approach was demonstrated by verifying the palmitoylation of the μ-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drug. The MOR has been reported to be palmitoylated using two other palmitoylation assays, but not BCC. Additionally, our optimized BCC protocol was able to measure changes in palmitoylation upon overexpression of two palmitoyl acyltransferases (PATs). This was the first demonstration that specific PATs are capable of affecting levels of palmitoylated MOR. While previous experiments in insect cells have shown that the D2R is palmitoylated, nothing was known about the site, function, or enzymes responsible. Here, the palmitoylation of the D2R was analyzed using our modified BCC protocol in a mammalian cell system. Analysis of a series of D2R mutations revealed that palmitoylation of the D2R occurs on the C-terminal cysteine residue (C443) of the polypeptide. Deletion of C443 led to an almost complete absence of D2R palmitoylation and significantly inhibited trafficking of the receptor to the plasma membrane. Rather, the C443 deletion mutant accumulated in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, the palmitoyl acyltransferase (PAT) zDHHC4 was identified as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 (GODZ) and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analysis of the D2R mutants indicates that palmitoylation of the receptor plays a critical role in stability but not the signaling capacity of the D2R.¬