The protease-activated receptors (PAR1 and PAR2) are unusual G protein-coupled receptors that are activated by unique serine proteases and are coexpressed in many different cell types. receptors stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but only PAR1 inhibited adenylyl cyclase activity, and pertussis toxin blocked PAR1 effects on both adenylyl cyclase and ERK1/2 signaling. Neu7 astrocytes express native PAR1 and PAR2 receptors that activate inositol phosphate, RhoA, and ERK1/2 signaling. However, only PAR1 inhibited adenylyl cyclase activity. PAR1 and PAR2 also stimulate Neu7 cell migration. PAR1 effects on ERK1/2 phosphorylation and cell migration were blocked both by pertussis toxin and by the mitogen-activated protein kinase kinase/ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126)], whereas PAR2 effects were only blocked by U0126. These studies demonstrate that PAR1 and PAR2 actually and functionally link to overlapping and unique information of G protein to differentially regulate downstream signaling pathways and cell physiology. Protease-activated receptors (PARs) are a family of four G protein-coupled receptors (GPCRs) that are irreversibly activated through proteolytic cleavage of their N termini by serine proteases (at the.g., thrombin, trypsin, plasmin, and others). This cleavage creates new extracellular N termini, which serve as tethered ligands that intramolecularly activate the receptors and initiate complex intracellular 112522-64-2 supplier signaling events (Macfarlane et al., 2001; Traynelis and Trejo, 2007). PAR1 was first discovered as a receptor for thrombin (Vu et al., 1991). As such, it is usually best known for its role in the cardiovascular system’s coagulation cascade and hemostatic mechanisms (Coughlin, 2005). A broader understanding of PAR1 and the cloning of three additional PARs (PAR2C4) (Nystedt et al., 1994; Ishihara et al., 1997; Xu et al., 1998) has implicated them in strikingly diverse pathophysiological functions, including stroke, inflammation, reactive gliosis, and malignancy (Ossovskaya and Bunnett, 2004). With regard to the role of PARs in stroke, mounting evidence implicates PAR1 and PAR2 in reactive gliosis after head injury and/or hemorrhagic stroke, which lead to the breakdown of the blood-brain hurdle of the central nervous system (CNS) (Traynelis and Trejo, 2007). Because PARs are expressed in both glia and neurons and in many other cells (Macfarlane et al., 2001; Ossovskaya and Bunnett, 2004), this leakage of serine proteases into the CNS provides PAR activators with direct access to their receptors after stroke and ischemia. PARs are believed to influence astrogliosis, which contributes to glial scarring and to the subsequent rebuilding of the blood-brain hurdle (Nishino et al., 1993; Pindon et al., 2000; Nicole et al., 2005). Conflicting reports have implicated PAR1 specifically in both neurodegeneration and neuroprotection, depending on the concentration of the activating protease (Traynelis and Trejo, 2007; Hamill et al., 2009). Whether these effects are more beneficial or harmful to recovering brain tissue remains unresolved. Furthermore, the molecular details underlying the function of PARs in these cells are not fully elucidated. PAR1 and PAR2 often are expressed in the same cells. In mediating their physiological effects, these closely related receptors have been reported to activate multiple G protein-linked signaling pathways, including mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and intracellular calcium (Dry et al., 1998; Macfarlane et al., 2001; Traynelis and Trejo, 2007). PAR1 seems to functionally couple to one or more of the Gq/11, Gi/o, and G12/13 subfamilies (Macfarlane et al., 2001; Traynelis and Trejo, 2007), and a previous screen for direct PAR1 binding partners found that Gi2 and Gq/11 both coimmunoprecipitate (coIP) with PAR1 in human neuroblastoma cells (Ogino et al., 1996). Several studies also have suggested that activating 112522-64-2 supplier PAR2 causes responses traditionally mediated by Gq/11, Gi/o, and G12/13 (Macfarlane et al., 2001; Traynelis and Trejo, 2007). However, a Rabbit Polyclonal to Shc (phospho-Tyr349) comprehensive understanding of the G protein-signaling pathways stimulated by PAR1 and PAR2 in the same cell is usually lacking. In the present study, we sought to define the G protein coupling and signaling information of PAR1 and PAR2 in the same cellular context and to identify differences in their physiological functions. Using both ectopic cellular systems conveying recombinant proteins (COS-7 kidney cells lacking functional PAR steps) and cells of neuronal source that natively express PARs (Neu7 astroglia), we have found that PAR1 and 112522-64-2 supplier PAR2 couple to overlapping and unique units of G proteins and linked signaling pathways to modulate different cellular responses. In doing so, we have highlighted previously unappreciated differences between these two closely related receptors. Materials and Methods Materials were obtained from the following sources: anti-FLAG M2 affinity solution and anti-FLAG M2 monoclonal antibody-peroxidase conjugate, bovine serum albumin.