Roles for a Cytoplasmic Tyrosine and Tyrosine Kinase Activity in the Interactions of Neu Receptors with Coated Pits

doi:10.1074/jbc.270.13.7061

Roles for a Cytoplasmic Tyrosine and Tyrosine Kinase Activity in the Interactions of Neu Receptors with Coated Pits (*)

From the (1)Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel and the
(2)Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

^§ To whom correspondence should be addressed. Tel.: 972-3-6409053; Fax: 972-3-6415053.

Abstract

The neu proto-oncogene product, p185 (HER2, c-ErbB-2), encodes a cell-surface tyrosine kinase receptor with high oncogenic potential, which correlates with increased tyrosine kinase activity and a rapid receptor internalization rate. To investigate the interactions and signal(s) leading to the endocytosis of Neu receptors, we employed lateral mobility and internalization studies. Fluorescence photobleaching recovery measurements revealed that activation of Neu receptors (induced by mutation or by agonistic antibodies) markedly reduced their mobile fractions. To elucidate the signals involved, other mutants, all carrying a constitutively dimerizing oncogenic mutation, were analyzed. A kinase-negative mutant and a mutant lacking all cytoplasmic tyrosine phosphorylation consensus sequences exhibited high mobile fractions, similar to nonactivated Neu. Retention of a single tyrosine autophosphorylation site (Tyr-1253) out of the five known such sites was sufficient to immobilize a large fraction of the receptor. For all mutants, internalization correlated with receptor immobilization and was blocked by treatments that interfere with coated pit structure, indicating that the immobilization is due to interactions with coated pits. This was supported by the coimmunoprecipitation of α-adaptin only with the constitutively activated Neu mutants. We conclude that activated Neu receptors become stably associated with coated pits via plasma membrane adaptor complexes (AP-2). Efficient Neu receptor endocytosis requires activation, a functional kinase domain, and at least one tyrosine autophosphorylation site.

Footnotes

↵* This work was supported in part by a project grant from the Israel Cancer Research Fund (to Y. I. H.) and by National Institutes of Health Grant CA51712 (to Y. Y.). 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:

EGF

epidermal growth factor

FPR

fluorescence photobleaching recovery

TMR

tetramethylrhodamine

HBSS

Hanks' balanced salt solution

BSA

bovine serum albumin

GAM

goat anti-mouse

SAM

sheep anti-mouse

PAGE

polyacrylamide gel electrophoresis

Mes

2-(N-morpholino)ethanesulfonic acid.
- Received November 4, 1994.