A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway.
Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4, 5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble -myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4, 5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.
Gaidarov
I
I
Department of Pharmacology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Chen
Q
Q
Falck
J R
JR
Reddy
K K
KK
Keen
J H
JH
D033942:Adaptor Proteins, Vesicular Transport; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D018699:Coated Vesicles / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004705:Endocytosis; D007295:Inositol Phosphates / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012995:Solubility
D033942:Adaptor Proteins, Vesicular Transport; D000345:Affinity Labels; D002966:Clathrin; D017931:DNA Primers; D007295:Inositol Phosphates; D008024:Ligands; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate