Protease-activated receptor-1 (PAR1) 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 1. Activated protein C (APC), an anti-coagulant protease, also activates PAR 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 1-deficient endothelial cells, indicating that PAR 1 is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR 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 1 in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR 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 1 in caveolae and a novel mechanism of PAR1 signal regulation.

Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. PARs play crucial roles in hemostasis and thrombosis, as well as in inflammation and vascular development. Coagulant proteases, which are generated at sites of vascular injury, act mainly through PARs to elicit signalling in a variety of cell types. Since PARs are irreversibly activated signalling must be tightly regulated. Desensitization and trafficking of proteolytically activated PARs control the magnitude, duration and spatial aspects of receptor signalling. Recent studies have revealed novel endocytic sorting mechanisms that regulate PAR signalling. PARs have also been implicated in tumor progression. PARs are overexpressed in several types of malignant cancer, transmit signals in response to tumor-generated proteases and promote tumor growth, invasion and metastasis. Recent work also indicates that matrix metalloprotease 1 (MMP-1) signals through PAR1 to promote tumor growth and invasion. In addition to PAR overexpression, tumor cells display aberrant PAR1 trafficking, which causes persistent signalling and cellular invasion. Thus, a novel type of gain-of-function in GPCR signalling in cancer can be acquired through dysregulation of receptor trafficking.

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signalregulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Ga i/o and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated ␣ subunit of heterotrimeric guanine nucleotide-binding G s protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G␣ s activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G␣ s and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.Prostate cancer, the most common noncutaneous malignant transformation in American men (1), starts as an androgen-dependent lesion in the prostate gland that can be successfully treated with surgical removal of the tumor or local radiation (2). Locally advanced and metastatic diseases are treated with endocrine therapies aimed to (i) decrease circulating androgen levels via chemical or physical castration, (ii) block androgen receptor (AR) 1 activation with anti-androgens, or (iii) achieve both (3). The hormonal therapies cause only a temporary shrinkage in tumor mass, and the cancer invariably reappears in the form of androgen-insensitive (AI) disease. Despite decades of intense basic and clinical research, to date there is no cure for AI prostate cancer.Accumulating evidence suggests that the AR itself is involved in the transition of prostate cancer from androgen-dependent to AI (4). AI prostate tumors express AR-regulated genes, including human kallikrein 2 and 3 (5), also known as prostate-specific antigen (PSA), suggesting that the AR is activated in these tumors despite the continued presence of the hormonal therapies. In vitro, the AR can be activated by factors other than androgens (6, 7), and in animal models increased AR expression was reported to be the only change associated with progression to hormone refractory disease (8). In humans, the AR is overexpressed in up to one-third of AI prostate carcinomas, suggesting that it could respond to low serum levels of androgens (9). Also, AI lesions exhibit frequent mutations in the AR that may allow it to be activated by other androgens or even anti-androgens (10). Hence, AR is a focal point in progression of the prostate cancer to advanced stage. However, the mechanisms regulating AR activation in the presence of low androgen concentrations and their significance to the progression to AI disease remain unclear.Transition of the prostate cancer to the AI stage is associated with increased expression of plasma membrane-localized G protein-coupled receptors (GPCRs) (11), which mediate cellular responses to a diverse array of extracellular molecules, including lipid and peptide growth factors (12). Human prostate cancer biopsies show elevated expression of the GPCRs for endothelin (13), bradykinin (14), and lysophosphatidic acid, 2 compared with benign specimens. The cancer...