Three-dimensional structure of the alpha1-beta complex in the skeletal muscle dihydropyridine receptor by single-particle electron microscopy.

The dihydropyridine receptor (DHPR) is a protein complex that consists of five distinct subunits of alpha(1), alpha(2), beta, gamma and delta and functions as a voltage-dependent L-type Ca(2+) channel. Here we purified the alpha(1)-beta complex (approximately 250 kDa) from the rabbit skeletal muscle DHPR and reconstructed its three-dimensional (3D) structure to 38 A resolution by single particle analysis of negative staining electron microscopy. The alpha(1)-beta structure exhibited two unique regions: a pseudo-4-fold petaloid region and an elongated region. X-ray crystallographic models of a homologous voltage-dependent K(+) channel and the beta subunit fit well into the individual regions of the alpha(1)-beta structure, revealing that the two regions correspond to the transmembrane alpha(1) and the cytoplasmic beta subunits, respectively. In addition, 3D reconstruction and immuno-electron microscopic analysis performed on the independently purified DHPR demonstrated that the alpha(1)-beta complex was located in the large globular portion of the DHPR, and the N-terminal region of the beta subunit was extended to the leg-shaped protrusion of the DHPR, which includes the alpha(2)delta subunits. Our results propose a model in which the beta subunit may regulate ion channel function by acting as a hinge between alpha(1) and alpha(2)delta subunits of the DHPR.

Combinatorial targeting of the macropinocytotic pathway in leukemia and lymphoma cells.

Ligand-directed delivery of agents to leukemia and lymphoma cells has the potential to yield new mechanistic disease insights and targeted therapies. Here we set out to target the macropinocytotic pathway with a combinatorial approach. From the screening of acute T-lymphoblastic leukemia Molt-4 cells with a random phage-display peptide library, we isolated a phage displaying the sequence CAYHRLRRC. This peptide contains a lymph node-homing motif (Cys-Ala-Tyr) and a cell-penetrating motif (Arg-Leu-Arg-Arg). Binding of this ligand-directed phage to a large panel of leukemia/lymphoma cells and to patient-derived samples was much higher than to non-leukemia control cells. CAYHRLRRC phage internalization into Molt-4 cells is both energy- and temperature-dependent. Flow cytometry with fluorescein-labeled peptide and endocytosis blocking with specific inhibitors revealed that CAYHRLRRC is indeed taken up through macropinocytosis in Molt-4 and K562 human leukemia cells. Unexpectedly, the cell surface receptor for the CAYHRLRRC peptide is not a heparan sulfate proteoglycan as it would be predicted for other cell-penetrating peptides. Confirming this interpretation, a CAYHRLRRC-directed peptidomimetic-induced cell death in all the leukemia and lymphoma cells was evaluated, whereas a control transactivator of transcription protein (tat)-directed proapoptotic peptidomimetic was non-selective. In summary, the targeting peptide CAYHRLRRC is selectively internalized through macropinocytosis in leukemia and lymphoma cells and has potential as a drug lead for ligand-directed anti-leukemia therapies.