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Updated: Jun 28, 2026
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Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins
Published on: March 29, 2020
Identification of G protein alpha subunit-palmitoylating enzyme
Ryouhei Tsutsumi1, Yuko Fukata, Jun Noritake
1Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
Molecular and Cellular Biology
|November 13, 2008
Summary
DHHC3 and DHHC7 enzymes palmitoylate G protein alpha subunits (Galpha), controlling their plasma membrane localization and G-protein-coupled receptor (GPCR) signaling. Their absence disrupts Galpha localization and blocks downstream signaling pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Heterotrimeric G protein alpha subunits (Galpha) are crucial for signal transduction, relaying signals from G-protein-coupled receptors (GPCRs) to cellular effectors.
- Galpha localization to the plasma membrane is regulated by reversible lipid palmitoylation.
Purpose of the Study:
- To identify the specific DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases responsible for Galpha palmitoylation.
- To elucidate the role of identified enzymes in Galpha localization, trafficking, and GPCR-mediated signaling.
Main Methods:
- Screening of 23 DHHC enzymes to identify Galpha palmitoylating enzymes.
- HEK293T cell culture, Galpha protein expression, and palmitoylation assays.
- Knockdown experiments using siRNA, Galpha relocalization studies (immunofluorescence microscopy), and photoconversion analysis.
- Fluorescence recovery after photobleaching (FRAP) to assess Galpha shuttling dynamics.
- Pharmacological stimulation of alpha(1A)-adrenergic receptor and assessment of downstream signaling.
Main Results:
- DHHC3 and DHHC7 were identified as key enzymes that robustly palmitoylate Galpha(q), Galpha(s), and Galpha(i2).
- Knockdown of DHHC3 and DHHC7 reduced Galpha(q/11) palmitoylation, caused its cytoplasmic relocalization, and impaired Galpha(q) shuttling between the plasma membrane and Golgi.
- DHHC3 and DHHC7 are essential for continuous Galpha(q) shuttling and blocking the alpha(1A)-adrenergic receptor/Galpha(q/11)-mediated signaling pathway.
Conclusions:
- DHHC3 and DHHC7 are critical regulators of Galpha protein localization and function.
- These enzymes control Galpha membrane targeting through palmitoylation, thereby modulating GPCR-mediated signal transduction.
- DHHC3 and DHHC7 are essential for maintaining the dynamic trafficking of Galpha proteins required for effective signaling.
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