RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes.

Intracellular pools of the heterotrimeric G-protein α-subunit Gαi3 (encoded by GNAI3) have been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was previously found to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas inhibitors of Gαi3, such as RGS4 and AGS3 (also known as GPSM1), markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38 (also known as TGOLN2)-labeled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-knockout mice resulted in a dramatic abrogation of autophagic flux, compared to wild-type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labeled vesicle pools are critical regulators of autophagic signaling that act via their ability to modulate phosphorylation of Bcl-2.

Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Stenosis of the superior limb of the systemic venous baffle following a Mustard procedure: an under-recognized problem.

BACKGROUND: Patients with atrioventricular concordance and ventriculoarterial discordance (DTGA) and a Mustard procedure may develop stenosis of the superior limb of the systemic venous baffle (SLSVB). The frequency of this complication in an adult cohort was evaluated. METHODS: Patients >18 years with DTGA and a Mustard procedure with and without a pacemaker (PM)/implantable cardioverter defibrillator (ICD) were identified through an institutional database. Subjects were included following a cardiac imaging study (computed tomography, magnetic resonance imaging, venography or cardiac catheterization) and follow-up in the PM/ICD or congenital cardiac clinics from 2001 to 2007. The primary end-point was narrowing of the SLSVB (<10mm) on cardiac imaging. Hemodynamically significant narrowing was defined by: azygous vein dilatation with retrograde flow or superior vena cava syndrome or the need for dilatation ± stenting of the SLSVB. RESULTS: Narrowing of the SLSVB was observed in 49/112 patients (70 males) age 31 ± 6 years (range 18-49) and was hemodynamically significant in 15/49. Of 29 patients with a PM (23) or ICD (6) and cardiac imaging, 17 had narrowing of the SLSVB which was hemodynamically significant in 8. Non-echocardiographic imaging had a sensitivity of 88% at detecting narrowing of the SLSVB in contrast to pulse-wave Doppler, which yielded a sensitivity of 16% (61% negative predictive value, 88% positive predictive value). CONCLUSIONS: In our adult cohort of Mustard patients, narrowing of the SLSVB had a prevalence of 44% and was more likely to be detected by non-echocardiographic imaging. Baffle patency should be evaluated before transvenous device implantation.