Protease-activated receptors (PAR) are G protein-coupled receptors that function as cell-surface sensors for coagulant proteases, as well as other proteases associated with the tumor microenvironment. PAR1 is activated by thrombin whereas the upstream coagulant protease VIIa bound to tissue factor and Xa can activate both PAR1 and PAR2. PAR1 has been implicated in tumor cell growth, migration, and invasion whereas the function of PAR2 in these processes is largely unknown. Towards defining the functional importance of PAR2 in cancer cells, we used small interfering RNAs to deplete highly invasive breast cancer cells of endogenous PAR proteins. Our findings strongly suggest that PAR2 is critical for MDA-MB-231 and BT549 breast cancer cell migration and invasion towards NIH 3T3 fibroblast conditioned medium. To define the relative importance of PAR1 versus PAR2 in mediating factor VIIa and Xa responses, we assessed signaling in cancer cells lacking either endogenous PAR1 or PAR2 proteins. Strikingly, in MDA-MB-231 cells depleted of PAR2, we observed a marked inhibition of VIIa and Xa signaling to phosphoinositide hydrolysis and extracellular signalregulated kinase 1/2 activation whereas signaling by VIIa and Xa remained intact in PAR1-deficient cells. Factor VIIa and Xa-induced cellular migration was also impaired in MDA-MB-231 cells deficient in PAR2 but not in cells lacking PAR1. Together, these studies reveal the novel findings that PAR2, a second protease-activated G protein-coupled receptor, has a critical role in breast cancer cell migration and invasion and functions as the endogenous receptor for coagulant proteases

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1 ⁄2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle ␣-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle ␣-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1 ⁄2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1 ⁄2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle ␣-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF cofactor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.

The irreversible proteolytic nature of protease-activated receptor-2 (PAR2) activation suggests that mechanism(s) responsible for termination of receptor signaling are critical determinants of the magnitude and duration of PAR2-elicited cellular responses. Rapid desensitization of activated G-protein-coupled receptors (GPCRs) involves both phosphorylation and binding of arrestins. Arrestins also function as scaffolds and transducers of mitogen-activated protein (MAP) kinase signaling cascades. The PAR2 cytoplasmic tail (C-tail) contains multiple sites of phosphorylation and may be an important determinant for arrestin interaction. Desensitization and internalization of activated PAR2 were markedly impaired in arrestin-deficient cells compared with wild-type control cells. PAR2 C-tail truncation mutants displayed normal agonist-induced internalization, caused rapid distribution of ␤arr2-GFP to the plasma membrane, and desensitized in an arrestin-dependent manner similar to that of wild-type PAR2. It is interesting that PAR2 C-tail mutants lost the capacity to stably associate with arrestins and consequently, redistributed to endocytic vesicles without ␤arr2-GFP, whereas internalized wild-type PAR2 remained stably associated with ␤arr2-GFP in endosomes. Moreover, activated PAR2 caused rapid and prolonged activation of endogenous extracellular signal-regulated kinase (ERK1/2). It was striking that in arrestin-deficient cells, activated PAR2 induced an initial peak in ERK1/2 activity that rapidly declined. The inability of internalized PAR2 C-tail mutants to stably associate with arrestins also resulted in loss of prolonged ERK2 activation. Thus, the PAR2 C-tail regulates the stability of arrestin interaction and kinetics of ERK1/2 activation but is not essential for desensitization or internalization. These findings further suggest that the diverse functions of arrestins in regulating PAR2 signaling and trafficking are controlled by multiple independent interactions involving both the intracellular loops and the C-tail.Protease-activated receptor-2 (PAR2) is activated by multiple trypsin-like serine proteases, including trypsin, tryptase, and coagulation proteases factors VIIa and Xa, but not by thrombin (Coughlin and Camerer, 2003). Because of the irreversible proteolytic nature of PAR2 activation and the generation of a tethered ligand that cannot diffuse away, mechanisms that contribute to the termination of signaling are critical determinants of the magnitude and kinetics of protease-elicited cellular responses. The regulation of PAR1 signaling has been extensively studied (Trejo, 2003), whereas considerably less is known about PAR2. G-protein-coupled receptors (GPCRs) are initially desensitized by rapid phosphorylation of agonist-occupied receptors by GPCR serine/ threonine kinases (GRKs) and other kinases (Krupnick and Benovic, 1998). In many cases, phosphorylation enhances receptor affinity for arrestin, and arrestin binding prevents receptor-G-protein interaction, thereby uncoupling the receptor fro...

Nucleotide‐activated P2Y1 receptors (P2Y1‐R) regulate a variety of physiological processes, including ion flux in epithelial cells. We utilized radioimmunoassays (RIA) and radioligand‐binding assays with [32P]MRS2500, a P2Y1‐R‐selective antagonist, to quantify agonist‐promoted internalization of the P2Y1‐R. Treatment of MDCK epithelial cells, which endogenously express the P2Y1‐R, with the P2Y1‐R‐selective agonist 2MeSADP resulted in a 40–50% decrease in cell‐surface [32P]MRS2500 binding sites with a t½ of ~12 min. Importantly, HA‐tagged human P2Y1‐R stably expressed in MDCK cells exhibited identical kinetics of agonist‐induced internalization. Deletion of residues 340–373 in the C‐terminus of the P2Y1‐R resulted in loss of agonist‐promoted internalization, and mutational analysis revealed that S352 and S354 were largely responsible for agonist‐promoted internalization. Interestingly, mutation of Ser336 and Thr339, which are predicted to be PKC phosphorylation sites, in the sequence S336RAT339 to Ala (ARAA) resulted in receptors that internalized faster (t1/2 = 4 min) and to a greater extent (70–80%) in response to agonist than wild‐type P2Y1‐R. In contrast, agonist‐promoted internalization was abolished when the SRAT sequence was mutated to DRAD. These data indicate that the C‐terminal tail is critical for both agonist‐induced endocytosis and plasma‐membrane‐retention of the P2Y1‐R.

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