Susanne Lutz

Structure of Gαq-p63RhoGEF-RhoA Complex Reveals a Pathway for the Activation of RhoA by GPCRs

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide–binding protein (G protein) Gαq and thereby links Gαq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Gαq-p63RhoGEF-RhoA complex, detailing the interactions of Gαq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Gαq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Gαq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

Protein Kinase D Selectively Targets Cardiac Troponin I and Regulates Myofilament Ca2+ Sensitivity in Ventricular Myocytes

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca2+]i transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca2+]i transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca2+]i transient amplitude. Complementary experiments in “skinned” myocytes confirmed reduced myofilament Ca2+ sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca2+ sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits SPECIFIC ACTIVATION OF Gsα BY AN NDPK B·Gβγ COMPLEX IN H10 CELLS

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of Gsα and Giα proteins. However, co-expression of Gsα led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to Gsα. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gβγ dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gβ subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B·Gβγ complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.

Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

OBJECTIVES This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. BACKGROUND Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. METHODS Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer-based assays, video edge detection, epifluorescence microscopy, and L-type Ca2(+) current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. RESULTS Myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2(+) current amplitude, and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. CONCLUSIONS PDE2 is markedly up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ1γ2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gαs and Gαi subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ1 orthologs, Gβ1 and Gβ1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ1/Gβ1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.

Regulation of Cardiac cAMP Synthesis and Contractility by Nucleoside Diphosphate Kinase B/G Protein βγ Dimer Complexes

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein &agr; subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein &bgr;&ggr;-dimers (G&bgr;&ggr;). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a G&bgr;1&ggr;2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the &bgr;1 subunit (G&bgr;1H266L&ggr;2). Recombinantly expressed G&bgr;1H266L&ggr;2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate G&bgr; phosphorylation. Compared with wild-type G&bgr;1&ggr;2 (G&bgr;1WT&ggr;2), overexpression of G&bgr;1H266L&ggr;2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/G&bgr;&ggr; complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing G&bgr;1H266L&ggr;2, the basal contractility was suppressed by ≈50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the &bgr;-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in G&bgr;1H266L&ggr;2 than in G&bgr;1WT&ggr;2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by G&bgr;1H266L&ggr;2. We conclude that the receptor-independent activation of G proteins via NDPK B/G&bgr;&ggr; complexes requires the intermediate phosphorylation of G protein &bgr; subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for G&bgr; and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

p63RhoGEF—a key mediator of angiotensin II-dependent signaling and processes in vascular smooth muscle cells

The purpose of our study was to investigate the role of endogenous p63RhoGEF in G(q/11)-dependent RhoA activation and signaling in rat aortic smooth muscle cells (RASMCs). Therefore, we studied the expression and subcellular localization in freshly isolated RASMCs and performed loss of function experiments to analyze its contribution to RhoGTPase activation and functional responses such as proliferation and contraction. By this, we could show that p63RhoGEF is endogenously expressed in RASMCs and acts there as the dominant mediator of the fast angiotensin II (ANG II)-dependent but not of the sphingosine-1-phosphate (S(1)P)-dependent RhoA activation. p63RhoGEF is not an activator of the concomitant Rac1 activation and functions independently of caveolae. The knockdown of endogenous p63RhoGEF significantly reduced the mitogenic response of ANG II, abolished ANG II-induced stress fiber formation and cell elongation in 2-D culture, and impaired the ANG II-driven contraction in a collagen-based 3-D model. In conclusion, our data provide for the first time evidence that p63RhoGEF is an important mediator of ANG II-dependent RhoA activation in RASMCs and therewith a leading actor in the subsequently triggered cellular processes, such as proliferation and contraction.

The function of rho-associated kinases ROCK1 and ROCK2 in the pathogenesis of cardiovascular disease

Rho-associated kinases ROCK1 and ROCK2 are serine/threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. ROCKs are involved in diverse cellular activities including actin cytoskeleton organization, cell adhesion and motility, proliferation and apoptosis, remodeling of the extracellular matrix and smooth muscle cell contraction. The role of ROCK1 and ROCK2 has long been considered to be similar; however, it is now clear that they do not always have the same functions. Moreover, depending on their subcellular localization, activation, and other environmental factors, ROCK signaling can have different effects on cellular function. With respect to the heart, findings in isoform-specific knockout mice argue for a role of ROCK1 and ROCK2 in the pathogenesis of cardiac fibrosis and cardiac hypertrophy, respectively. Increased ROCK activity could play a pivotal role in processes leading to cardiovascular diseases such as hypertension, pulmonary hypertension, angina pectoris, vasospastic angina, heart failure, and stroke, and thus ROCK activity is a potential new biomarker for heart disease. Pharmacological ROCK inhibition reduces the enhanced ROCK activity in patients, accompanied with a measurable improvement in medical condition. In this review, we focus on recent findings regarding ROCK signaling in the pathogenesis of cardiovascular disease, with a special focus on differences between ROCK1 and ROCK2 function.

p63RhoGEF and GEFT are Rho-specific guanine nucleotide exchange factors encoded by the same gene

Activation of Rho GTPases, which play pivotal roles in diverse cellular functions, is catalysed by specific guanine nucleotide exchange factors (GEFs). We and others (Souchet et al. (2002)) independently cloned a human cDNA encoding a 580 aa protein (p63RhoGEF), which contains a tandem of Dbl homology and pleckstrin homology domains typical for RhoGEFs. In accordance with Souchet et al., recombinant p63RhoGEF interacted with and catalysed GDP/GTP exchange at RhoA, but not Rac1 or Cdc42. Recently, an N-terminally truncated form of p63RhoGEF, termed GEFT, was described as a Rac/Cdc42-specific GEF (Guo et al. 2003). As judged by RT-PCR with specific primers, we were able to detect mRNA variants encoding p63RhoGEF and GEFT within several tissues and cell lines. Apparently, they co-exist within one cell and are derived from the same gene. When expressed in human embryonic kidney cells, both p63RhoGEF and GEFT caused activation of RhoA, but not Rac1 or Cdc42, and induced serum response factor-mediated gene transcription, which was fully blunted by the Rho-inactivating C3 transferase. In line with these data, expression of either p63RhoGEF or GEFT in J82 human bladder carcinoma cells induced the formation of actin stress fibres. We therefore conclude that p63RhoGEF and GEFT are apparently isoforms derived from the same gene and that GEFT, similar to p63RhoGEF, activates RhoA in several cell types.

Angiotensin II modulates VEGF-driven angiogenesis by opposing effects of type 1 and type 2 receptor stimulation in the microvascular endothelium

Vascular endothelial growth factor (VEGF) is a main stimulator of pathological vessel formation. Nevertheless, increasing evidence suggests that Angiotensin II (Ang II) can play an augmentory role in this process. We thus analyzed the contribution of the two Ang II receptor types, AT(1)R and AT(2)R, in a mouse model of VEGF-driven angiogenesis, i.e. oxygen-induced proliferative retinopathy. Application of the AT(1)R antagonist telmisartan but not the AT(2)R antagonist PD123,319 largely attenuated the pathological response. A direct effect of Ang II on endothelial cells (EC) was analyzed by assessing angiogenic responses in primary bovine retinal and immortalized rat microvascular EC. Selective stimulation of the AT(1)R by Ang II in the presence of PD123,319 revealed a pro-angiogenic activity which further increased VEGF-driven EC sprouting and migration. In contrast, selective stimulation of the AT(2)R by either CGP42112A or Ang II in the presence of telmisartan inhibited the VEGF-driven angiogenic response. Using specific inhibitors (pertussis toxin, RGS proteins, kinase inhibitors) we identified G(12/13) and G(i) dependent signaling pathways as the mediators of the AT(1)R-induced angiogenesis and the AT(2)R-induced inhibition, respectively. As AT(1)R and AT(2)R stimulation displays opposing effects on the activity of the monomeric GTPase RhoA and pro-angiogenic responses to Ang II and VEGF requires activation of Rho-dependent kinase (ROCK), we conclude that the opposing effects of the Ang II receptors on VEGF-driven angiogenesis converge on the regulation of activity of RhoA-ROCK-dependent EC migration.