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

• Published in: The Journal of biological chemistry Vol. 283; no. 12; pp. 8034 - 8045
• Main Authors: Wang, Ping-Yuan, Weng, Jian, Lee, Sungsoo, Anderson, Richard G.W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.03.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; HeLa Cells; Humans; Hydroxycholesterols - metabolism; MAP Kinase Signaling System - physiology; Mitogen-Activated Protein Kinase 1 - genetics; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - genetics; Mitogen-Activated Protein Kinase 3 - metabolism; Mutation, Missense; Peptide Mapping; Protein Binding - genetics; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Protein Structure, Tertiary; Protein Tyrosine Phosphatases, Non-Receptor - genetics; Protein Tyrosine Phosphatases, Non-Receptor - metabolism; Rabbits; Receptors, Steroid - genetics; Receptors, Steroid - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M707631200

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 OSBP408-809, 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.