A Functional Phosphatidylinositol 3,4,5-Trisphosphate/Phosphoinositide Binding Domain in the Clathrin Adaptor AP-2 α Subunit. IMPLICATIONS FOR THE ENDOCYTIC PATHWAY

doi:10.1074/jbc.271.34.20922

A Functional Phosphatidylinositol 3,4,5-Trisphosphate/Phosphoinositide Binding Domain in the Clathrin Adaptor AP-2 α Subunit. IMPLICATIONS FOR THE ENDOCYTIC PATHWAY*

From the ^‡ Department of Pharmacology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the
^§ University of Texas Southwestern Medical Center, Dallas, Texas 75235

^¶ To whom correspondence should be addressed:
Dept. of Pharmacology and Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., BLSB/915, Philadelphia, PA 19107.
Tel.: 215-503-4624; Fax: 215-923-1098; E-mail: keen{at}lac.jci.tju.edu.

Abstract

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 D-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 α 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 α sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian α, 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.

Footnotes

↵* This work was supported by the Robert A. Welch Foundation (to J. R. F.) and by Grant GM-49217 from the National Institutes of Health (to J. H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

AP

assembly or associated proteins

PPI

polyphosphoinositols and polyphosphoinositides

PIP₃

phosphatidylinositol 3,4,5-trisphosphate

PI-3-kinases

phosphatidylinositol 3-kinases

diC₈

di-octanoyl

MES

2-(N-morpholino)ethanesulfonic acid

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

PAGE

polyacrylamide gel electrophoresis

IPTG

isopropylthiogalactoside

8-Az-ATP

8-Azido-adenosine-5′-triphosphate

PEG

polyethylene glycol

IP₆

D-myo-inositol hexakisphosphate

IP₄

D-myo-inositol 1,3,4,5-tetraphosphate

IP₃

D-myo-inositol 1,4,5-triphosphate

IP

inositol phosphate.
↵²O. B. Goodman, J. G. Krupnick, F. Santini, V. V. Gurevich, R. B. Penn, A. W. Gagnon, J. H. Keen, and J. L. Benovic, submitted for publication.
- Received April 18, 1996.
- Revision received June 11, 1996.