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J. Biol. Chem., Vol. 283, Issue 12, 8034-8045, March 21, 2008

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The N Terminus Controls Sterol Binding while the C Terminus Regulates the Scaffolding Function of OSBP^*

From the Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9039

Previously we reported that when cell cholesterol is acutely lowered with β-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP_408-809, it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.

Received for publication, September 11, 2007 , and in revised form, December 6, 2007.

^* This work was supported in part by Grants HL 20948 and GM 52016 from the National Institutes of Health, the Cecil H, Green Distinguished Chair in Cellular and Molecular Biology, and the Perot Family Foundation (all to R. G. W. A.) and the American Heart Association 0730122N (to P.-Y. W.). 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 supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed: Dept. of Cell Biology, University of Texas Southwestern Medical Center Dallas, TX 75390-9039. Tel.: 214-648-2346; Fax: 214-648-7577; E-mail: richard.anderson{at}utsouthwestern.edu.

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