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 hex akisphosphate (InsP(6)) (K-d = 239 nM) and diphosphoinositol pentakisp hosphate (PP-InsP(5)) (K-d = 22 nM). The specificity of this ligand bi nding was demonstrated by competitive displacement of bound [H-3]InsP( 6). IC50 values were as follows: PP-InsP(5) = 50 nM, InsP(6) = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P-5) = 2.2 mu M, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P-5) = 5 mu M, ino sitol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P-4) > 10 mu M, inositol- 1,4,5-trisphosphate (Ins(1,4,5)P-3) > 10 mu M. Moreover, 10 mu M inosi tol hexasulfate (InsS(6)) displaced only 15% of [H-3]InsP(6). The phys iological significance of this binding is the ligand-specific inhibiti on of clathrin assembly (PP-InsP(5) > InsP(6) > Ins(1,2,4,5,6)P-5); In s(1,3,4,5,6)P-5 and InsS(6) did not inhibit clathrin assembly. We also observed high affinity binding of InsP(6) to purified bovine brain AP -3. We separately expressed the 33-kDa amino terminus and the 58-kDa c arboxyl terminus, and it was the former that contained the high affini ty inositol polyphosphate binding site, These studies suggest that spe cific inositol polyphosphates may play a role in the regulation of syn aptic function by interacting with the synapse-specific clathrin assem bly protein AP-3.