Phosphatidylinositol 3-Kinase Activity Is Required at a Postendocytic Step in Platelet-derived Growth Factor Receptor Trafficking

doi:10.1074/jbc.270.22.13225

Phosphatidylinositol 3-Kinase Activity Is Required at a Postendocytic Step in Platelet-derived Growth Factor Receptor Trafficking (*)

From the (1) Program in Molecular Medicine and the Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and the
National Jewish Hospital for Immunology and Respiratory Medicine, Denver, Colorado 80206

^§ To whom correspondence should be addressed:
Dept. of Cell Biology, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605.
Tel.: 508-856-6898; Fax: 508-856-4289; E-mail: SCorvera{at}BANGATE1.UMMED.EDU.

Abstract

We have previously reported that platelet-derived growth factor (PDGF) receptor mutants that lack high affinity binding sites for phosphatidylinositol 3-kinase (PI 3-kinase) fail to concentrate in juxtanuclear vesicular structures after activation with PDGF. We have now identified the point in the endocytic pathway at which PI 3-kinase binding sites are required. Receptor internalization from the plasma membrane, measured as the acquisition of acid resistance of prebound I-PDGF, was only slightly decreased in cells expressing a PDGF receptor mutant (F5) lacking PI 3-kinase, GTPase-activating protein (GAP), phospholipase C, and Syp binding sites but not expressing mutants where any of these individual sites were restored nor expressing a mutant lacking exclusively PI 3-kinase binding sites. In contrast, the extent of down-regulation of PDGF binding sites from the cell surface after prolonged incubation with PDGF as well as the degradation of [S]methionine-labeled receptor were markedly reduced in cells expressing the F5 mutant, mutants restored in GAP, phospholipase C, or Syp binding sites or expressing the mutant exclusively lacking PI 3-kinase binding sites but not in cells expressing the mutant where PI 3-kinase binding sites were restored. Inhibition of PI 3-kinase activity with wortmannin caused a dramatic decrease in the rates of down-regulation and degradation of wild-type receptors. These results suggest that PI 3-kinase binding sites are not required for internalization of PDGF receptor but are required to divert the PDGF receptor to a degradative pathway. Furthermore, the requirement for PI 3-kinase binding sites on the receptor appears to be due to a requirement for PI 3-kinase catalytic activity.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK-40330 (to S. C.) and GM48339 (to A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PDGF

platelet-derived growth factor

GAP

GTPase-activating protein

PI 3-kinase

phosphatidylinositol 3-kinase

DMEM

Dulbecco's modified Eagle's medium

EGF

epidermal growth factor.
↵²The mutants used in this study are designated in the following way. The receptor in which tyrosines at positions 740, 751, 771, 1009, and 1021 were replaced by phenylalanine is called F5. The “add-back” mutants represent F5 receptors in which phenylalanines at positions 740, 751, 771, 1009, or 1021 were replaced by tyrosine individually or in combination and are designated by the single letter code for tyrosine, followed by the position number of the replaced residue. The add-back mutant in which phenylalanines at positions 740 and 751 were replaced by tyrosine is called Y40/51. The wild-type receptor in which tyrosines at positions 740 and 751 were replaced by phenylalanine is called F40/51.