Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation.
We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs.
Program in Chemical Biology, Penn State College of Medicine, Hershey, PA 17033, USA; Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA. Electronic address: bae154@psu.edu.
Brittany
B
Ebersole
Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.
Jessica
J
Petko
Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.
Robert
R
Levenson
D000906:Antibodies / Q000276:immunology; D057930:Click Chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D018189:Immunomagnetic Separation; D047468:Immunoprecipitation; D054878:Lipoylation; D017450:Receptors, Opioid, mu / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000906:Antibodies; D005231:Fatty Acids, Unsaturated; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; C047007:15-hexadecynoic acid
Click chemistry; Immunoprecipitation; Mu-opioid receptor; Palmitoylation