Photoisomerization of a protein bound chromophore is the basis of the light sensing and signaling responses of many photoreceptors. Z-to-E photoisomerization of the Pr Cph1Δ2 phytochrome has been investigated by polarization resolved femtosecond visible pump-infrared probe spectroscopy, which yields structural information on the Pr excited (Pr*), Pr ground, and lumi-R product states. By exhaustive search analysis, two photoreaction time constants of (4.7 ± 1.4) and (30 ± 5) ps were found. Ring D orientational change in the electronic excited state to the transition state (90° twist) has been followed in real-time. Rotation of ring D takes place in the electronically excited state with a time constant of 30 ± 5 ps. The photoisomerization is best explained by a single rotation around C(15)═C(16) methine bridge in the Pr* state and a diffusive interaction with its protein surrounding.

The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis. Apolipoprotein E receptor 2 or apoER2 (also called LRP8) is a member of the low-density lipoprotein receptor (LDL-R) family and binds ligands including apolipoproteins E (apoE), apoB, apoJ and reelin (1-4). It is expressed predominantly in the brain, platelets, ovary, epididymis and placenta (1,5,6). Apolipoprotein E receptor 2 shares several structural characteristics with LDL-R, including an extracellular domain composed of modules of ligand-binding repeats, a single transmembrane domain and a cytoplasmic domain containing one NPXY motif, which is determinant for the clathrin-mediated endocytosis of LDL-R (1,7). As a receptor for reelin, apoER2 participates in brain development and synaptic plasticity (2,8,9). Upon reelin ligand binding, apoER2 induces the activation of Src family kinases and the phosphorylation of disabled 1 (Dab1), which binds to the NPXY motif and initiates a signaling cascade including the activation of phosphatidylinositol-3-kinase and the inhibition of glycogen synthase kinase-3b (9). Apolipoprotein E receptor 2 also has a role in the LDL-dependent sensitization of platelets by inducing the activation of p38MAPK (4). One of the interesting characteristics of apoER2 is its expression in several spliced variants (10-12). These structural variations differ in the receptor's ligand-bindi...