This review will focus on the role of the activation segment in the mediation of these different aspects of and David J. Owen control. The activation segment is defined as the region Laboratory of Molecular Biophysics spanning conserved sequences DFG and APE and cor-and Oxford Centre for Molecular Sciences responds to residues 184-208 in cAPK (Taylor and Rad-University of Oxford zio-Andzelm, 1994). The activation segment includes Oxford OX1 3QUThr-197, one of two autophosphorylation sites in cAPK. United KingdomThe conversion of an inactive kinase to an active kinase involves conformational changes in the protein that lead

hours after transfection. COS-7 cells were incubated with EGF (0.1 g/ml) [biotinylated, complexed to Texas Red-streptoavidin (Molecular Probes, Eugene, OR)] in binding buffer [20 mM Hepes-NaOH ( pH 7.5), 130 mM NaCl, and 0.1% bovine serum albumin] at 4°C for 60 min. Internalization of EGF was allowed by incubation in Dulbecco's modified Eagle's medium at 37°C for 10 min, then excess EGF was removed with 0.2 M AcOH ( pH 2.5) and 0.5 M NaCl at 4°C for 5 min. Cells were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with a polyclonal antibody to myc (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein isothiocyanate-conjugated antibody to rabbit (Organon Teknika, Boxtel, Netherlands). Internalization of EGF was observed by confocal microscopy (Bio-Rad). 37. We thank Y. Watanabe (Ehime University, Japan) for providing us with various synthetic phosphoinositides.

AP2 is the best-characterized member of the family of heterotetrameric clathrin adaptor complexes that play pivotal roles in many vesicle trafficking pathways within the cell. AP2 functions in clathrin-mediated endocytosis, the process whereby cargo enters the endosomal system from the plasma membrane. We describe the structure of the 200 kDa AP2 "core" (alpha trunk, beta2 trunk, mu2, and sigma2) complexed with the polyphosphatidylinositol headgroup mimic inositolhexakisphosphate at 2.6 A resolution. Two potential polyphosphatidylinositide binding sites are observed, one on alpha and one on mu2. The binding site for Yxxphi endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered mu2 linker, is necessary to allow Yxxphi motif binding. A model for AP2 recruitment and activation is proposed.