Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels.
The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsPn (BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPn analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O-acyl-linked PtdInsPn analogs that interacted with proteins penetrating the bilayer. The InsPn and PtdInsPn probes bind with highest selectivity and affinity to the two alpha subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa mu subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPn binding site of alpha-AP-2 prefers more highly phosphorylated species, and (iii) the protein-acyl chain interactions showed high capacity but low selectivity.
Department of Chemistry, University at Stony Brook, Stony Brook, New York 11794-3400, USA.
A A
AA
Profit
J
J
Chen
Q M
QM
Gu
A
A
Chaudhary
K
K
Prasad
E M
EM
Lafer
G D
GD
Prestwich
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D015335:Molecular Probes / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D013388:Succinimides / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D001577:Benzophenones; D002966:Clathrin; D007295:Inositol Phosphates; D008024:Ligands; D015335:Molecular Probes; C096731:N-succinimidyl 4-benzoyldihydrocinnamate; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D019920:Photoaffinity Labels; D013388:Succinimides