Protease-activated receptor-1 in human brain: localization and functional expression in astrocytes $ Candice E. Junge, a C. Justin Lee, a Katherine B. Hubbard, a Zhoabin Zhang, b Jeffrey J. Olson, b John R. Hepler, a Daniel J. Brat, c and Stephen F. Traynelis a, * a Department of Pharmacology, Emory University, Atlanta, GA 30322, USA b Department of Neurosurgery, Emory University, Atlanta, GA 30322, USA c Department of Pathology, Emory University, Atlanta, GA 30322, USA Received 14 November 2003; revised 11 February 2004; accepted 23 February 2004 Available online 10 May 2004 Abstract Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca 2+ , indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca 2+ . Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders. D 2004 Elsevier Inc. All rights reserved. Keywords: Protease-activated receptor; PAR1; Thrombin; G-protein coupled receptor; Serine protease; Glioblastoma; Astrocyte Introduction Protease-activated receptor-1 (PAR1) is a member of a family of four unique G-protein coupled receptors, PAR1– 4. PAR1 is well known for its actions in clotting and hemostasis (Coughlin, 2001; Macfarlane et al., 2001), but the role of PAR1 and its activators, serine proteases, in the central nervous system (CNS) has received increased atten- tion in recent years. PAR1 is widely expressed throughout the rodent CNS in both neurons and glia and is upregulated following experimental ischemia in hippocampal slice cul- tures (Niclou et al., 1998; Rohatgi et al., 2003; Striggow et al., 2001; Weinstein et al., 1995). Protease-activated recep- tors have a novel mode of activation in which serine proteases cleave the extracellular N-terminal of the receptor and unmask a peptide sequence that acts as a tethered ligand to constitutively activate the receptor. Thrombin is the most potent activator of PAR1 (Vu et al., 1991). Other serine proteases including Factor Xa, the Factor VIIa/tissue factor complex, and streptokinase complexed with plasminogen can activate PAR1 (Camerer et al., 2000, 2002; McRed- mond et al., 2000; Riewald et al., 2001), whereas plasmin and trypsin both activate and inactivate the receptor (Junge 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.02.018 $ Grant Support: NIH-NS 39419 (SFT), NIH-NS 42505 (CEJ), NIH- NS36147 (DJB), NIH-NS43875 (CJL), NIH-NS37112 and GM61847 (JRH), and NARSAD (SFT). * Corresponding author. Department of Pharmacology, Rollins Re- search Center, Emory University School of Medicine, 1510 Clifton Road, Room 5025, Atlanta, GA 30322. Fax: +1-404-727-0365. E-mail address: strayne@emory.edu (S.F. Traynelis). www.elsevier.com/locate/yexnr Experimental Neurology 188 (2004) 94 – 103