Phospholipase C⑀ (PLC⑀) is a novel class of phosphoinositide-specific PLC characterized by possession of CDC25 homology and Ras/Rap1-associating domains. We and others have shown that human PLC⑀ is translocated from the cytoplasm to the plasma membrane and activated by direct association with Ras at its Ras/Rap1-associating domain. In addition, translocation to the perinuclear region was induced upon association with Rap1⅐GTP. However, the function of the CDC25 homology domain remains to be clarified. Here we show that the CDC25 homology domain of PLC⑀ functions as a guanine nucleotide exchange factor for Rap1 but not for any other Ras family GTPases examined including Rap2 and Ha-Ras. Consistent with this, coexpression of fulllength PLC⑀ or its N-terminal fragment carrying the CDC25 homology domain causes an increase of the intracellular level of Rap1⅐GTP. Concurrently, stimulation of the downstream kinases B-Raf and extracellular signalregulated kinase is observed, whereas the intracellular level of Ras⅐GTP and Raf-1 kinase activity are unaffected. In wild-type Rap1-overexpressing cells, epidermal growth factor induces translocation of PLC⑀ to the perinuclear compartments such as the Golgi apparatus, which is sustained for at least 20 min. In contrast, PLC⑀ lacking the CDC25 domain translocates to the perinuclear compartments only transiently. Further, the formation of Rap1⅐GTP upon epidermal growth factor stimulation exhibits a prolonged time course in cells expressing fulllength PLC⑀ compared with those expressing PLC⑀ lacking the CDC25 homology domain. These results suggest a pivotal role of the CDC25 homology domain in amplifying Rap1-dependent signal transduction, including the activation of PLC⑀ itself, at specific subcellular locations such as the Golgi apparatus.

No abstract

Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation.

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.Ras proteins are small guanine nucleotide-binding proteins that serve as molecular switches in regulation of cellular proliferation and differentiation by cycling between the active GTP-bound and the inactive GDP-bound forms (for a review, see Ref. 1). In mammalian cells, the GTP-bound Ras exerts its action by physically associating with and activating effector proteins, such as the serine/threonine kinase Raf-1, through its effector region (amino acid residues 32-40 in human Ha-Ras). In addition to Raf-1 and its isoforms B-Raf and A-Raf, recent searches have identified a number of Ras effectors (or effector candidates) that associate directly with Ras in a GTP-dependent manner (for a review, see Ref.2). Two of them, RalGDS 1

BACKGROUND Excessive binge alcohol drinking has acute cardiac arrhythmogenic effects, including promotion of atrial fibrillation (AF), which underlies “Holiday Heart Syndrome.” The mechanism that couples binge alcohol abuse with AF susceptibility remains unclear. We previously reported stress-activated c-Jun N-terminal kinase (JNK) signaling contributes to AF development. This is interesting because JNK is implicated in alcohol-caused organ malfunction beyond the heart. OBJECTIVES The purpose of this study was to detail how JNK promotes binge alcohol-evoked susceptibility to AF. METHODS The authors found binge alcohol-exposure leads to activated JNK, specifically JNK2. Furthermore, binge alcohol induces AF (24- vs. 1.8-Hz burst pacing-induced episodes per attempt per animal), higher incidence of diastolic intracellular Ca 2+ activity (Ca 2+ waves, sarcoplasmic reticulum [SR] Ca 2+ leakage), and membrane voltage (V m ) and systolic Ca 2+ release spatiotemporal heterogeneity (Δt Vm-Ca ). These changes were completely eliminated by JNK inhibition both in vivo and in vitro. calmodulin kinase II (CaMKII) is a proarrhythmic molecule known to drive SR Ca 2+ mishandling. RESULTS The authors report for the first time that binge alcohol activates JNK2, which subsequently phosphorylates the CaMKII protein, enhancing CaMKII-driven SR Ca 2+ mishandling. CaMKII inhibition eliminates binge alcohol-evoked arrhythmic activities. CONCLUSIONS Our studies demonstrate that binge alcohol exposure activates JNK2 in atria, which then drives CaMKII activation, prompting aberrant Ca 2+ waves and, thus, enhanced susceptibility to atrial arrhythmia. Our results reveal a previously unrecognized form of alcohol-driven kinase-on-kinase proarrhythmic crosstalk. Atrial JNK2 function represents a potential novel therapeutic target to treat and/or prevent AF.

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein ␤2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the G␤ 1 subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that G␤␥ subunits enhance the activity of these enzymes provided either G␣ s or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of G␤␥ subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by G␤␥ subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous G␤␥ subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G i was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, G␤␥ subunits both in vitro and in intact cells. Moreover, G␤␥ subunits derived from a source(s) other than G i are necessary for the full activation of hACVI by isoproterenol in intact cells.

BackgroundRecently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown.Methods and ResultsWe show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries.Conclusionsα1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.

No abstract