Regulation of AP-3 Function by Inositides

doi:10.1074/jbc.272.10.6393

Regulation of AP-3 Function by Inositides

IDENTIFICATION OF PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE AS A POTENT LIGAND*

From the ^‡ Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, the
^§ Inositol Lipid Section, Laboratory of Signal Transduction, NIEHS, Research Triangle Park, North Carolina 27709, the
^¶ Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas, 75235, and the
^∥ Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112

** To whom correspondence should be addressed:
Dept. of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245.
Tel.: 210-567-7220; Fax: 210-567-7247; E-mail: Lafer{at}uthscsa.edu

Abstract

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC₈PtdIns(3,4,5)P₃) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[³H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC₈-PtdIns(3,4,5)P₃ was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC₈-PtdIns(3,4,5)P₃ that establish specificity. Dioctanoyl-phosphatidylinositol 3,4-bisphosphate, which does not have a 5-phosphate, and 4,5-O-bisphosphoryl-D-myo-inosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC₈-PtdIns(3,4,5)P₃ as inhibitors of clathrin assembly. Deacylation of DiC₈-PtdIns(3,4,5)P₃ reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.

Footnotes

↵* This work was supported by National Institutes of Health NINDS Grant NS29051 (to E. M. L.), NIGMS Grant GM31278 (to J. R. F.), and NINDS Grant NS29632 (to G. D. P.), and by NEN Life Sciences. 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:

PtdIns(3,4,5)P₃

phosphatidylinositol 3,4,5-trisphosphate

AP

assembly protein

InsP₆

D-myo-inositol hexakisphosphate

Ins(1,4,5)P₃

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

PtdIns(4,5)P₂

phosphatidylinositol 4,5-bisphosphate

DiC₁₁-O-PtdIns(4,5)P₂

4,5-O-bisphosphoryl-D-myo-inosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate

Gro

L-α-glycero

DiC₈

dioctanoyl

PtdIns(3,4)P₂

phosphatidylinositol 3,4-bisphosphate

GST

glutathione S-transferase

InsS₆

myo-inositol hexasulfate

MES

2-(N-morpholino)ethanesulfonic acid.
- Received September 12, 1996.
- Revision received December 27, 1996.