Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

BACKGROUND: Two pertussis toxin sensitive G(i) proteins, G(i2) and G(i3), are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i) isoforms are functionally distinct. To test for isoform-specific functions of G(i) proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). METHODS: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2) (Gα(i2) (-/-)) or Gα(i3) (Gα(i3) (-/-)). mRNA levels of Gα(i/o) isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i) and Ca(v)α(1) protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. RESULTS: In cardiac tissue from Gα(i2) (-/-) mice, Gα(i3) mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3) (-/-) mouse hearts, Gα(i2) mRNA (127 ± 5%) and protein (131 ± 10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2) (-/-) mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα(i3) (-/-) mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2) (but not of Gα(i3)) and following treatment with pertussis toxin in Gα(i3) (-/-). The pore forming Ca(v)α(1) protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(v)α(1) and Ca(v)β(2) subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2). CONCLUSION: Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα(i) proteins. In particular, loss of Gα(i2) is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway

Diverging gain-of-function mechanisms of two novel KRAS mutations associated with Noonan and cardio-facio-cutaneous syndromes

Activating somatic and germline mutations of closely related RAS genes (H, K, N) have been found in various types of cancer and in patients with developmental disorders, respectively. The involvement of the RAS signalling pathways in developmental disorders has recently emerged as one of the most important drivers in RAS research. In the present study, we investigated the biochemical and cell biological properties of two novel missense KRAS mutations (Y71H and K147E). Both mutations affect residues that are highly conserved within the RAS family. KRASY71H showed no clear differences to KRASwt, except for an increased binding affinity for its major effector, the RAF1 kinase. Consistent with this finding, even though we detected similar levels of active KRASY71H when compared with wild-type protein, we observed an increased activation of MEK1/2, irrespective of the stimulation conditions. In contrast, KRASK147E exhibited a tremendous increase in nucleotide dissociation generating a self-activating RAS protein that can act independently of upstream signals. As a consequence, levels of active KRASK147E were strongly increased regardless of serum stimulation and similar to the oncogenic KRASG12V. In spite of this, KRASK147E downstream signalling did not reach the level triggered by oncogenic KRASG12V, especially because KRASK147E was downregulated by RASGAP and moreover exhibited a 2-fold lower affinity for RAF kinase. Here, our findings clearly emphasize that individual RAS mutations, despite being associated with comparable phenotypes of developmental disorders in patients, can cause remarkably diverse biochemical effects with a common outcome, namely a rather moderate gain-of-function

Transforming acidic coiled coil 1 promotes transformation and mammary tumorigenesis

1. The journal Cancer Research is the original source of the material.2. This article is hosted on a website external to the CBCRA Open Access Archive. Selecting “View/Open” below will launch the full-text article in another browser window

Better understanding of phosphoinositide 3-Kinase (PI3K) pathways in vasculature: towards precision therapy targeting angiogenesis and tumor blood supply

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3K{alpha} isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3K{alpha} and other class I isoforms, i.e. PI3K{beta}, {gamma}, {delta}, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3K{gamma} and PI3K{delta} (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100 nM) and LY294002 (10 {my}M). These vasoconstrictions were also inhibited by the PI3K{alpha} isoform inhibitors A66 (10 {my}M) and PI-103 (1 {my}M), but not by the PI3K{beta} isoform inhibitor TGX 221 (100 nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3K{alpha} is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via {alpha}1-adrenergic receptors. Our results highlight a regulatory role of PI3K{alpha} in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3K{alpha} in cancer cells and tumor angiogenesis and regional blood flow

Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin.

Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs