Structural study of membrane proteins (GPCR and channels) regulating signal transduction.

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The following research results were achieved in three years by support of Glant-in Aid for Specially promoted Research from 2001. 1)Structure of water channel, AQP4, was analyzed by over expression technique of Sf9 cells as well as electron crystallography. Based on the structure we could elucidate mechanisms of orthogonal array formation and cell adhesion of the water channel molecules. 2)Structure and gating mechanism of the acetylcholine receptor pore were analyzed by cryo-electron microscopy (J.Mol.Biol., 319, 1165-1176 (2002), Nature, 423, 949-955 (2003)). 3)A new computer program for automatic particle pickup method of single particle analysis, by which structure of voltage-sensitive sodium channel was analyzed (Nature, 409, 1047-1051 (2001)), was developed (J.Struct.Biol., 136, 227-238 (2001)) and enabled us to analyze structure of IP_3 receptor (J.Mol.Biol., 336, 155-164 (2004)). 4)Structure of Homer was analyzed by X-ray crystallography (J.Mol.Biol., 318, 1117-1126 (2002)), and we developed a new Co-localization Expression Technique (Co-LET) for purification of membrane proteins without detergents (BBRC, 295, 756-765 (2002)). 5)We studied interaction of endothelin B receptor with caveoline-1 (Eur.J.Biochem., 270, 1816-1827 (2003)). Structure of rhodopsin was analyzed at 2.6 Å (Proc.Natl.Acad.Sci.USA, 99, 5982-5987 (2002)). The mutations of three residues in connexin26 were studied to obtain the structural insights of the molecule (J.Boil.Chem., 278, 1807-1816 (2003)). The importance of the second PDZ domain for clustering of PSD-95 was revealed (J.Biol.Chem., 277, 3640-3646 (2002)).