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Protease-activated receptor-1 ([PAR.sub.1]) is a G-protein-coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and endothelial barrier permeability through the activation of [PAR.sub.1]. Activated protein C (APC), an anti-coagulant protease, also activates [PAR.sub.1]. However, unlike thrombin, APC elicits anti-inflammatory responses and protects against endothelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in [PAR.sub.1]-deficient endothelial cells, indicating that [PAR.sub.1] is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by [PAR.sub.1], we examined the effect of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate endothelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a marked increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on endothelial barrier integrity. Strikingly, APC signaling and endothelial barrier protection effects were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of [PAR.sub.1] in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces [PAR.sub.1] phosphorylation and desensitizes endothelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC exposure. Thus, APC selective signaling and endothelial barrier protective effects are mediated through compartmentalization of [PAR.sub.1] in caveolae and a novel mechanism of [PAR.sub.1] signal regulation. APC | endothelial | GPCR | Rac1 | thrombin

We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, G[H.sub.4][C.sub.1], with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by Rac, but signaling was restored by expression of a toxin-insensitive mutant of PP5, Y451A, which we engineered. PP5 is unique among protein phosphatases in that it contains an N-terminal regulatory domain with three tetratricopeptide repeats (TPR) that inhibit its activity. Expression of the TPR domain coupled to GFP blocked channel stimulation by the thyroid hormone. We also show that the published structures of the PP5 TPR domain and the TPR domain of p67, the Rac-binding subunit of NADPH oxidase, superimpose over 92 [alpha] carbons. Mutation of the PP5 TPR domain at two predicted contact points with Rac-GTP prevents the TPR domain from functioning as a dominant negative and blocks the ability of Y451A to rescue signaling in the presence of okadaic acid. PP5 stimulation by Rac provides a unique molecular mechanism for the antagonism of Rho-dependent signaling through protein kinases in many cellular processes, including metastasis, immune cell chemotaxis, and neuronal development. KCNH2 | tetratricopeptide repeat | neuronal development | potassium channel | thyroid hormone

Many nuclear hormones have physiological effects that are too rapid to be explained by changes in gene expression and are often attributed to unidentified or novel G protein-coupled receptors. Thyroid hormone is essential for normal human brain development, but the molecular mechanisms responsible for its effects remain to be identified. Here, we present direct molecular evidence for potassium channel stimulation in a rat pituitary cell line (GH4C1) by a nuclear receptor for thyroid hormone, TR[beta], acting rapidly at the plasma membrane through phosphatidylinositol 3-kinase (PI3K) to slow the deactivation of KCNH2 channels already in the membrane. Signaling was disrupted by heterologous expression of TR[beta] receptors with mutations in the ligand-binding domain that are associated with neurological disorders in humans, but not by mutations that disrupt DNA binding. More importantly, PI3Kdependent signaling was reconstituted in cell-free patches of membrane from CHO cells by heterologous expression of human KCNH2 channels and TR[beta], but not TR[alpha], receptors. TR[beta] signaling through PI3K provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism. neuronal development | phosphatidylinositol 3-kinase | potassium channels | Rac | KCNH2