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J. Biol. Chem., Vol. 278, Issue 43, 42679-42685, October 24, 2003

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Functional Regulation of Tissue Plasminogen Activator on the Surface of Vascular Smooth Muscle Cells by the Type-II Transmembrane Protein p63 (CKAP4)^*

Tahir M. Razzaq, Rosemary Bass, David J. Vines, Finn Werner¶, Simon A. Whawell||, and Vincent Ellis**

From the School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom

We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with ¹²⁵I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.

Received for publication, May 30, 2003 , and in revised form, July 10, 2003.

^* This work was funded by British Heart Foundation Grants PG/96041 and PG/98172. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a sequence comparison of rat and human p63 (CKAP4).

Present address: Dept. of Experimental Haematology, The Royal London Hospital Medical School, London E1 2AD, United Kingdom.

Present address: Ludwig Inst. for Cancer Research, University College London, London W1W 7BS, United Kingdom.

^¶ Present address: Dept. of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

^|| Present address: Eastman Dental Inst., University College London, London WC1X 8LD, United Kingdom.

^** Senior Research Fellow of the British Heart Foundation and to whom correspondence should be addressed. Tel.: 44-1603-592570; Fax: 44-1603-592250; E-mail: v.ellis{at}uea.ac.uk.

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