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) (K_d = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP₅) (K_d = 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 μM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P₅) = 5 μM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,S)P₄) > 10 μM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) > 10 μM. Moreover, 10 μM 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.