Inhibition of clathrin assembly by high affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3.
Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.
Ye
W
W
Center for Molecular Medicine, University of Texas Health Science Center, San Antonio 78245.
Ali
N
N
Bembenek
M E
ME
Shears
S B
SB
Lafer
E M
EM
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D017931:DNA Primers; D007295:Inositol Phosphates / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D034041:Monomeric Clathrin Assembly Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013379:Substrate Specificity; D013569:Synapses / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D017931:DNA Primers; D007295:Inositol Phosphates; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; D010833:Phytic Acid