Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110γ.

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.

Engineering A-kinase anchoring protein (AKAP)-selective regulatory subunits of protein kinase A (PKA) through structure-based phage selection.

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.

Formin-dependent synaptic growth: evidence that Dlar signals via Diaphanous to modulate synaptic actin and dynamic pioneer microtubules.

The diaphanous gene is the founding member of a family of Diaphanous-related formin proteins (DRFs). We identified diaphanous in a screen for genes that are necessary for the normal growth and stabilization of the Drosophila neuromuscular junction (NMJ). Here, we demonstrate that diaphanous mutations perturb synaptic growth at the NMJ. Diaphanous protein is present both presynaptically and postsynaptically. However, genetic rescue experiments in combination with additional genetic interaction experiments support the conclusion that dia is necessary presynaptically for normal NMJ growth. We then document defects in both the actin and microtubule cytoskeletons in dia mutant nerve terminals. In so doing, we define and characterize a population of dynamic pioneer microtubules within the NMJ that are distinct from the bundled core of microtubules identified by the MAP1b-like protein Futsch. Defects in both synaptic actin and dynamic pioneer microtubules are correlated with impaired synaptic growth in dia mutants. Finally, we present genetic evidence that Dia functions downstream of the presynaptic receptor tyrosine phosphatase Dlar and the Rho-type GEF (guanine nucleotide exchange factor) trio to control NMJ growth. Based on the established function of DRFs as Rho-GTPase-dependent regulators of the cell cytoskeleton, we propose a model in which Diaphanous links receptor tyrosine phosphatase signaling at the plasma membrane to growth-dependent modulation of the synaptic actin and microtubule cytoskeletons.

A genetic screen for mouse mutations with defects in serotonin responsiveness.

The serotonergic system plays a key role in regulating basic behaviors. Deficits in serotonergic neurotransmission have been implicated in psychiatric disorders, such as schizophrenia and depression. Here we have optimized a behavioral screen and performed a small scale genetic screen to identify genes involved in serotonin responsiveness in the mouse. Treatment of mice with serotonin, serotonin precursors, or serotonin agonists results in a quantifiable head twitch response (HTR), which is drug dosage-dependent and dependent on the 5-HT2A receptor system. This assay can uncover variation in serotonin responsiveness as shown by our identification of inbred strains with high, medium, and low head twitch responses to administration of the serotonin agonist DOI (+-1-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane). We chose C57Bl/6J mice for our mutagenesis screen, because of their robust HTR and because of the availability of their complete genomic sequence. We optimized this assay by examining dose and age dependence of DOI-induced HTR in 6-week and 3-month-old C57BL/6J mice. HTR decreases only slightly in 3-month-old mice, and a substantial but submaximal HTR is induced by 0.75-1 mg/kg of DOI. We assayed HTR in response to DOI of 247 G1 C57BL/6J progeny from C57BL/6J males, which had been mutagenized with ethylnitrososurea (ENU), and recovered one provisionally heritable hyper-responsive mutation. This and future mutations recovered via this protocol may provide ideal subjects for the study of human psychiatric disorders, such as depression and schizophrenia, and thereby aid in the development of better therapeutic strategies for these disorders. Thus, it is well worth expanding on this genetic screen in its current form and by addition of further pharmacologic assays in the future.

Signal integration through blending, bolstering and bifurcating of intracellular information.

A cell's response to its environment is often determined by signaling through the actions of enzyme cascades. The ability to organize these enzymes into multiprotein complexes allows for a high degree of fidelity, efficiency and spatial precision in signaling responses.