Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

PLOS ONE, Dec 2019

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate 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, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 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 analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

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Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

November Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability Brittany Ebersole 0 1 2 Jessica Petko 0 1 2 Matthew Woll 0 1 2 Shoko Murakami 0 1 2 Kate Sokolina 0 1 2 Victoria Wong 0 1 2 Igor Stagljar 0 1 2 Bernhard Lüscher 0 1 2 Robert Levenson 0 1 2 0 Current address: Science Department, The Pennsylvania State University , York, Pennsylvania , United States of America 1 1 Department of Pharmacology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, United States of America, 2 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 3 Department of Biochemistry, University of Toronto , Toronto, Ontario , Canada , 4 Department of Molecular Genetics, University of Toronto , Toronto, Ontario , Canada , 5 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 6 Center for Molecular Investigation of Neurological Disorders, The Pennsylvania State University , University Park, Pennsylvania , United States of America 2 Editor: Adriano Marchese, Loyola University Chicago, Stritch School of Medicine, UNITED STATES We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate 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, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 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 analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Palmitoylation is a posttranslational modification that can affect protein stability, membrane association, subcellular trafficking, desensitization, internalization, and signaling of a variety of proteins including G protein-coupled receptors (GPCRs; [1–7]). Palmitoylation typically involves covalent attachment of palmitate (saturated 16-carbon fatty acid) to proteins via an Competing Interests: The authors have declared that no competing interests exist. enzymatic reaction that is catalyzed, in humans, by a family of 23 known palmitoyl acyltransferases (PATs; [8]). PATs consist of four to six transmembrane domains and share a conserved aspartic acid-histidine-histidine-cysteine (DHHC) domain [9–14]. Palmitoylation enhances the hydrophobicity of proteins, thereby contributing to their membrane association, proteinprotein interactions, and subcellular trafficking [15–17]. In the present study, we used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method [18, 19], to interrogate the palmitoylation status of the D2 dopamine receptor (D2R). D2Rs are GPCRs which, upon activation, couple to Gi proteins and lead to an inhibition of adenylate cyclase [18]. D2Rs are alternatively spliced and exist as two isoforms. The long isoform (D2L) contains a 29 amino acid insert in the third intracellular loop that is absent in the short isoform (D2S) [20–22]. This region is believed to be involved in specificity of G protein coupling. D2Rs are involved in a variety of cellular pathways, including those responsible for cognition, emotion, reward, and motor control [23, 24]. Dysfunction of D2R-mediated cellular signaling is associated with a number of human disorders including Parkinson’s disease, schizophrenia, and drug abuse [25]. The D2R is of particular pharmacological interest as it is the target for many antipsychotic drugs [26–28]. Previous studies, using baculovirus-infected insect Sf9 cells, have shown that the human D2Rs are palmitoylated [29, 30]. While no experimental information is currently available regarding the structural or functional significance of D2R palmitoylation in mammalian cells, a recent simulation study suggests that, like most Class A GPCRs, the D2R is palmitoylated on a cysteine residue at the terminus of helix 8 ( (...truncated)


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Brittany Ebersole, Jessica Petko, Matthew Woll, Shoko Murakami, Kate Sokolina, Victoria Wong, Igor Stagljar, Bernhard Lüscher, Robert Levenson. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability, PLOS ONE, 2015, Volume 10, Issue 11, DOI: 10.1371/journal.pone.0140661