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

PLoS One. 2015 Nov 4;10(11):e0140661. doi: 10.1371/journal.pone.0140661. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / metabolism
  • Amino Acid Sequence
  • Cell Membrane / metabolism
  • Click Chemistry
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Golgi Apparatus
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Lipoylation
  • Membrane Proteins / metabolism
  • Microscopy, Confocal
  • Protein Stability
  • Receptors, Dopamine D2 / chemistry*
  • Receptors, Dopamine D2 / genetics
  • Receptors, Dopamine D2 / metabolism
  • Sequence Alignment
  • Two-Hybrid System Techniques

Substances

  • Membrane Proteins
  • Receptors, Dopamine D2
  • Acyltransferases
  • ZDHHC3 protein, human
  • ZDHHC8 protein, human
  • Cysteine