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Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (TH2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks TH2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell-specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

AKAPs (A-kinase–anchoring proteins) act as scaffold proteins that anchor the regulatory subunits of the cAMP-dependent PKA (protein kinase A) to coordinate and compartmentalize signaling elements and signals downstream of Gs-coupled GPCRs (G protein–coupled receptors). The β2AR (β-2-adrenoceptor), as well as the Gs-coupled EP2 and EP4 (E-prostanoid) receptor subtypes of the EP receptor subfamily, are effective regulators of multiple airway smooth muscle (ASM) cell functions whose dysregulation contributes to asthma pathobiology. Here, we identify specific roles of the AKAPs Ezrin and Gravin in differentially regulating PKA substrates downstream of the β2AR, EP2R (EP2 receptor) and EP4R. Knockdown of Ezrin, Gravin, or both in primary human ASM cells caused differential phosphorylation of the PKA substrates VASP (vasodilator-stimulated phosphoprotein) and HSP20 (heat shock protein 20). Ezrin knockdown, as well as combined Ezrin and Gravin knockdown, significantly reduced the induction of phospho-VASP and phospho-HSP20 by β2AR, EP2R, and EP4R agonists. Gravin knockdown inhibited the induction of phospho-HSP20 by β2AR, EP2R, and EP4R agonists. Knockdown of Ezrin, Gravin, or both also attenuated histamine-induced phosphorylation of MLC20. Moreover, knockdown of Ezrin, Gravin, or both suppressed the inhibitory effects of Gs-coupled receptor agonists on cell migration in ASM cells. These findings demonstrate the role of AKAPs in regulating Gs-coupled GPCR signaling and function in ASM and suggest the therapeutic utility of targeting specific AKAP family members in the management of asthma. [ABSTRACT FROM AUTHOR]

Cyclic ADP‐ribose (cADPR) has emerged as a calcium‐regulating second messenger in smooth muscle cells. CD38 protein possesses ADP‐ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP‐ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET‐1 elicited [Ca2+]i responses, and 8‐Br‐cADPR, a cADPR antagonist significantly attenuated agonist‐induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium. [ABSTRACT FROM AUTHOR]

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.