DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

Abstract

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α(1A)-adrenergic receptor-stimulated intracellular Ca(2+) mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability.

Figure 1

Cys2 is critical for oxidation-sensitive proteasome degradation of RGS4 protein in MDA-MB-231 cells (A) and HEK293 cells (B). Cells were transfected with 1 μg plasmids encoding HA-tagged WT RGS4 or RGS4C2S mutant. Cells under hypoxia (+) or nomoxia (-) for 24 hrs were treated without or with 20 μM MG132 for 4 hrs. Proteins in cell lysate were subjected to western blot analysis with anti-HA and anti-β-actin antibodies. Images shown are representatives of four separate experiments.

Figure 2

Co-expression of active DHHC proteins increases expression levels of RGS4 but not RGS4C2S mutant. HEK293 cells were transfected with HA-tagged WT RGS4 or RGS4C2S mutant in the absence or presence of Myc-tagged DHHC3, DHHC7 or their inactive C157S or C160S mutants. Proteins in cell lysate were analyzed by western blot using anti-HA, anti-Myc and anti-β-actin antibodies. Images shown are representatives of three separate experiments. (A). Co-expression of DHHC3 or DHHC7 only caused WT RGS4 to accumulate in cells and migrate more rapidly during electrophoresis (B). Acyltransferase activity is critical for DHHC3 and DHHC7 to modulate RGS4.

Figure 3

DHHC3 palmitoylates RGS4 in HEK293 cells. Cell lysates were analyzed by western blot. Images are representatives of 3–6 separate experiments. (A). DHHC3 increased RGS4 protein in a dose-dependent manner, which was blocked by 2-BP (100 μM). (B). DTT or hydroxylamine reversed DHHC3-induced increase in the mobility of RGS4. (C). HEK293 cells co-transfected with GFP-tagged DHHC3 and RGS4 or RGS4C2S were metabolically labeled with 17-ODYA. RGS4 and RGS4C2S were immunoprecipitated and biotin labeled. Samples were treated with Tris-HCl (lanes 1–3) or hydroxylamine (lanes 4–6) for 1 hr prior to western blot analysis using anti-HA antibody and streptavidin-IRDye800. (D). DHHC3 forms complex with RGS4 but not RGS4C2S in HEK293 cells. Expression of DHHC3-Myc was analyzed by western blot (upper panel). HA-tagged RGS4, RGS4C2S, and associated DHHC3-Myc were immunoprecipitated using anti-HA affinity agarose beads and detected by western blot using anti-HA and anti-Myc antibodies, respectively (lower panel).

Figure 4

Palmitoylation protects RGS4 from oxidation-sensitive degradation. HEK293 cells were transfected with RGS4C2S, RGS4 or RGS4 and DHHC3, cultured under hypoxia for 24 hrs and then transferred to normoxia in the presence of cycloheximide (100 μM). Oxidation-induced degradation of pre-accumulated RGS4 or its mutant was examined over time by western blot assays. (A). Representative western blot. MG132 blocked RGS4 degradation. (B). Time-course of RGS4 degradation obtained from three independent experiments. RGS4 protein was quantified by densitometry and initial optical density units in each group were set as 1. Points, mean; bars, SE.

Figure 5

Palmitoylated RGS4 attenuates α_1A-AR-stimulated intracellular Ca²⁺ mobilization. HEK293 cells stably expressing α_1A-AR were loaded with Flur-4. Elevations in intracellular Ca²⁺ were quantified by the ratio of phenylephrine-induced peak fluorescence vs. maximum fluorescence in the presence of A23187 (2 μM). (A). Phenylephrine concentration-response curve in the absence (Control) or presence of prazosin (1 μM) (n=4). (B). Co-expression of RGS4 with DHHC3 attenuated phenylephrine-stimulated elevation in intracellular Ca²⁺. Data are typical of three experiments.