title: Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.
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.
authors.affiliation: Department of Pharmacology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, United States of America.
authors.forename: Brittany
authors.initials: B
authors.lastname: Ebersole
authors.affiliation: Department of Pharmacology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, United States of America.
authors.forename: Jessica
authors.initials: J
authors.lastname: Petko
authors.affiliation: Department of Pharmacology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, United States of America.
authors.forename: Matthew
authors.initials: M
authors.lastname: Woll
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Shoko
authors.initials: S
authors.lastname: Murakami
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Kate
authors.initials: K
authors.lastname: Sokolina
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Victoria
authors.initials: V
authors.lastname: Wong
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
authors.affiliation: Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.|Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.|Center for Molecular Investigation of Neurological Disorders, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Lüscher
authors.affiliation: Department of Pharmacology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Levenson
mesh_terms: D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D057930:Click Chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D054878:Lipoylation; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D055550:Protein Stability; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
chemical_list: D008565:Membrane Proteins; D017448:Receptors, Dopamine D2; D000217:Acyltransferases; C574550:ZDHHC3 protein, human; C488097:ZDHHC8 protein, human; D003545:Cysteine