Identification of G Protein α Subunit-Palmitoylating Enzyme

Abstract

The heterotrimeric G protein ␣ subunit (G␣) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as G␣ palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated G␣ q , G␣ s , and G␣ i2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased G␣ q/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that G␣ q rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous G␣ q shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the ␣ 1A -adrenergic receptor/G␣ q/11 -mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling G␣ localization to the plasma membrane.G-protein-coupled receptors (GPCRs) form the largest family of cell surface receptors, consisting of more than 700 members in humans. GPCRs respond to a variety of extracellular signals, including hormones and neurotransmitters, and are involved in various physiologic processes, such as smooth muscle contraction and synaptic transmission (20,25). Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, transduce signals from GPCRs to their effectors and play a central role in the GPCR signaling pathway (13,21,24,32). Although the G␣ subunit seems to localize stably at the cytosolic face of the plasma membrane (PM), a recent report suggested that G␣ o , a G␣ isoform, shuttles rapidly between the PM and intracellular membranes (2). The PM targeting of G␣ requires both interaction with the G␤␥ complex and subsequent lipid palmitoylation of G␣ (22). Thus, palmitoylation of G␣ is a critical determinant of membrane targeting of the heterotrimer G␣␤␥.Protein palmitoylation is a common posttranslational modification with lipid palmitate and regulates protein trafficking and function (7,18). G␣ is a classic and representative palmitoyl substrate (19,38), and recent studies revealed that protein palmitoylation modifies virtually almost all the components of G-protein signaling, including GPCRs, G␣ subunits, several members of the RGS (regulators of G-protein signaling) family of GTPase-activating proteins, GPCR kinase GRK6, and some small GTPases (7, 33). This common lipid modification plays an important role in compartmentalizing G-protein signaling to the specific microdomain, such as membrane caveolae and lipid raft (26). The palmitoyl thioester bond is relatively labile, and palmitates on substrates turn over rapidly, allowing proteins to shuttle between the cytoplasm/intracellular organelles and the PM (2, 3, 27). For example, binding of isoproterenol to the ␤-adrenergic receptor markedly accelerates the depalmitoylation of the a...

Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

“…To do so, we applied a strategy (overexpression then siRNA) set up by Fukata et al 14,28,29 and used by several for the identification of DHHC/substrate pairs. Among the 23 DHHC proteins, overexpression of 5 of them, namely DHHC1, 11, 3, 7 and 14 is able to enhance Fas palmitoylation.…”

In Silico Screening for Palmitoyl Substrates Reveals a Role for DHHC1/3/10 (zDHHC1/3/11)-mediated Neurochondrin Palmitoylation in Its Targeting to Rab5-positive Endosomes

“…In contrast to DHHC1/10 (z1/11), the DHHC3/7 subfamily is exclusively localized at the Golgi apparatus (Fig. 7C) (29,30). Given that DHHC1 and -10 (z11) represent palmitoylating enzymes associated with early endosomes, DHHC1 and/or 10 (z11) may locally palmitoylate Ncdn and thereby actively recruit Ncdn to early endosomal membranes.…”

“…The following cDNAs were kindly provided by indicated investigators: human Rab6A, Franck Perez (Institut Curie); rat CaMKII␣, Ulli Bayer (University of Colorado, Denver, CO); human TRPC1, Yasuo Mori (Kyoto University); mouse TRPM8, Ardem Patapoutian (The Scripps Research Institute, La Jolla, CA), and Makoto Tominaga (National Institute for Physiological Sciences, Japan); mouse orexin 2 receptor, Akihiro Yamanaka (Nagoya University); rat TARP ␥-2, rat TARP ␥-8, and human liprin-␣2, David Bredt (Johnson and Johnson Research and Development, San Diego, CA); rat KIF5C (amino acids 1-560), Gary Banker (Oregon Health and Science University, Portland, OR); rat homer 1C, Akihiko Kato (Eli Lilly, Indianapolis, IN); rat kalirin7, Xin-Ming Ma (University of Connecticut Health Science Center, Farmington, CT). Other constructs used in this study were described previously (9,29,30).…”

“…7, A and B). Interestingly, the DHHC1/10 (z1/11) subfamily, but not the Golgi-resident DHHC3 (29,30), was co-localized with Rab5-positive endosomes in dendrites (Fig. 7C).…”

“…Because of the availability of the specific antibodies, only TARP ␥-8, CaMKII␣, Ncdn, and (Fig. 3, A-C) (9,30). Palmitoylation of all four substrates was enhanced by DHHC3 and -7, which belong to the same subfamily (Fig.…”