FIG. 9.
DHHC3 and -7 are necessary for GPCR-mediated signal transduction through the Gαq palmitoylation. (A) α1A-AR activation leads to CREB phosphorylation. HeLa cells transiently transfected with α1A-AR were treated with 50 μM phenylephrine with or without 50 μM prazosin for 5 min. The cell lysates were treated with 10% trichloroacetic acid, and the resulting precipitates were subjected to immunoblotting with antibodies to CREB and Ser133 pCREB. (B to D) HeLa cells were transiently transfected with α1A-AR and indicated plasmids or siRNAs. (B) α1A-AR-mediated CREB phosphorylation requires palmitoylated Gαq. Knockdown (KD) of Gαq and Gα11 blocked α1A-AR-induced CREB phosphorylation, and RNAi-resistant Gαq (WT), but not palmitoylation-deficient Gαq (CS), rescued α1A-AR-mediated CREB phosphorylation. (C) Knocked down DHHC3 and DHHC7 (DHHC3/7 KD) inhibited α1A-AR-induced CREB phosphorylation, indicating that DHHC3 and -7 are essential for the GPCR-signaling pathway through Gαq palmitoylation. pCREB/CREB ratios are indicated in the graph. Error bars show ±SD (n = 3). **, P < 0.01. (D) HeLa cells expressing α1A-AR were treated with phenylephrine and stained with anti-pCREB antibody (red). Note that α1A-AR stimulation induced robust phosphorylation of CREB in the nucleus. The defect of CREB phosphorylation by knocked down human DHHC3 and human DHHC7 (hDHHC3/7 KD) was rescued by GFP-mDHHC3 (WT; green) but not catalytically inactive GFP-mDHHC3 (C157S) [mDHHC3 (CS)]. Scale bar, 20 μm. (E) The HeLa cells cotransfected with α1A-AR and the indicated siRNAs were treated with phenylephrine for 10 min, followed by IP3 extraction. Average IP3 production of three independent experiments is indicated in the graph. Agonist-induced IP3 production was specifically blocked by knockdown of DHHC3 and -7 (KD 3/7), indicating that DHHC3 and -7 are necessary for the α1A-AR-mediated GPCR signaling pathway. Error bars show ±SD (n = 3). **, P < 0.01; n.s., not significant (P = 0.20).