G protein betagamma subunits have potential as a target for therapeutic treatment of number diseases. We performed virtual docking small-molecule library to site on Gbetagamma that mediates interactions. hypothesized differential targeting this surface could allow selective modulation subunit functions. Several compounds bound with affinities from 0.1 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways HL-60...

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)ϵ that plays critical role in maximal β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) cardiac myocytes. Here demonstrate PLCϵ phosphatidylinositol 4,5-bisphosphate hydrolytic activity PLCϵ-stimulated Rap1 GEF are both required for PLCϵ-mediated enhancement sarcoplasmic reticulum significantly enhances Rap activation response to βAR the heart. Downstream activity,...

Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) cardiac myocytes. Here have reported for the first time a pathway downstream betaAR involving cAMP-dependent Rap GTP exchange factor, Epac, and regulates CICR To demonstrate Epac CICR, myocytes were treated with Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased...

Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca 2+ ) signals in response to various external cues. Notably, IP R channel activity is determined several obligatory factors, including , Ca and ATP. The critical basic amino acid residues the N-terminal -binding core (IBC) region that facilitate binding are well characterized. In contrast, conferring regulation have yet be ascertained. Using...

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, the putative phosphorylation sites that modulate its function are reflected as changes communication. Growth factor stimulation has been correlated with decrease communication parallel activation of ERK1/2; inhibition epidermal growth (EGF)-induced Cx43 uncoupling was observed by using MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated factors,...

Calcium can be mobilized in pancreatic β-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR by activating both protein kinase A Epac2. Here we provide the first report that novel phosphoinositide-specific phospholipase C- (PLC-) is expressed islets Langerhans, knockout (KO) PLC- gene expression mice disrupts action to these mice. Thus, present study, which wild-type (WT) C57BL/6 mouse were loaded with photolabile...

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks sarcoplasmic reticulum (SR) cisternae and extensions transverse-tubules (TT) that increase co-localization proteins required for store-operated Ca2+ entry (SOCE). Here, we report SOCE, peak transient amplitude force production during repetitive stimulation are increased after exercise parallel with time course TT association SR-stacks. Unexpectedly, also activated constitutive coincident a modest...

Loss of endoplasmic reticular (ER) Ca2+ activates store-operated entry (SOCE) by causing the ER localized sensor STIM to unfurl domains that activate Orai channels in plasma membrane at contact sites (MCS). Here, we demonstrate a novel mechanism which inositol 1,4,5 trisphosphate receptor (IP3R), an ER-localized IP3-gated channel, regulates neuronal SOCE. In human neurons, SOCE evoked pharmacological depletion ER-Ca2+ is attenuated loss IP3Rs, and restored expression IP3Rs even when they...

Phospholipase Cepsilon (PLCepsilon) has been suggested to transduce signals from small GTPases, but its biological function not yet clarified. Using astrocytes PLCepsilon-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLCepsilon. Stimulation by lysophospholipids occurs G(i), whereas activates PLC Rho. Further studies reveal PLCepsilon is...

The plasma membrane lipid PI4P is the main source of phosphoinositides generated by GPCR activation.

We previously developed peptides that bind to G protein βγ subunits and selectively block interactions between a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, Gross, R. A., Taussig, R., Smrcka, A. V. (2001) EMBO 20, 767–776). Here, we created cell-permeating versions some these by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated βγ-binding...

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by specific isoform phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation perinuclear PLCε is not clear. Here we tested role G protein βγ subunits. Gβγ inhibition blocked ET-1-stimulated PI4P depletion in neonatal and adult ventricular myocytes. Blocking inhibited...

Adenylyl cyclase (AC) converts ATP into cyclic AMP (cAMP), an important second messenger in cell signaling. Heterotrimeric G proteins and other regulators are for control of AC activity. Depending on the isoform, G<i>βγ</i> subunits can either conditionally stimulate or inhibit cAMP synthesis. We previously showed that G<i>α</i>_s-<i>βγ</i> heterotrimer binds to N terminus (NT) type 5 (AC5). now show NT a wide variety isoforms. hypothesized G<i>βγ</i>/AC5 interactions involving...

Opposing effects of G i protein α and βγ subunits on the abundance second messenger cAMP enable neutrophil migration.

We previously identified a 10-amino acid region from the Y domain of phospholipase Cbeta2 (PLCbeta2) that associates with G-protein betagamma subunits (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154). mapped site for cross-linking synthetic peptide (N20K) corresponding to this Cys(25) within amino-terminal coiled-coil Gbetagamma (Yoshikawa, D. M., Bresciano, K., Hatwar, (2001) 276, 11246-11251). Here, further experiments series variable length...

Ryanodine receptor type I (RYR1)-related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype childhood onset non-dystrophic muscle disorders. There no treatments for this severe diseases. A major barrier to therapy development is lack an animal model mirrors clinical severity pediatric cases disease. To address this, we used CRISPR/Cas9 gene editing generate novel recessive mouse RYR1 RM. This (Ryr1TM/Indel)...

Duchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a influx mechanism coordinated STIM1 sensors of luminal within sarcoplasmic reticulum (SR) Ca2+-permeable Orai1 channels sarcolemma, proposed to contribute Ca2+-mediated damage DMD. To directly determine impact Orai1-dependent SOCE on dystrophic phenotype, we crossed mdx...

Big mitogen-activated protein kinase 1/extracellular-regulated 5 (BMK1/ERK5) is regulated sequentially by a series of upstream MAP kinases (MEKs) in signaling cascade. MEKs activate their downstream MAPK phosphorylation threonine and tyrosine the T- X-Y motif. MEK5 BMK1 exists as naturally occurring splice variants, MEK5α MEK5β. The full-length (MEK5α) 89 amino acids longer than MEK5β at N terminus, but precise functional difference between two variants not known. Dual site mutation (Ser-311...

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, perturbation as-sociated signals contribute to pathogenesis severity remains largely uncharacterized....

Peptides derived from a random-peptide phage display screen with purified Gβ₁γ₂ subunits as the target promote dissociation of G protein heterotrimers in vitro and activate signaling intact cells. In vitro, one these peptides (SIRKALNILGYPDYD; SIRK) promotes subunit by binding directly to Gβγ accelerating GαGDP without catalyzing nucleotide exchange. The experiments described here were designed test whether mechanism SIRK action is fact We created mutant Gβ₁...