Subsets of Epidermal Growth Factor Receptors during Activation and Endocytosis

doi:10.1074/jbc.272.7.4079

Subsets of Epidermal Growth Factor Receptors during Activation and Endocytosis*

From the Department of^‡ Pharmacology and the
^§ Department of Microbiology and Immunology, Kimmel Cancer Institute, Philadelphia, Pennsylvania 19107

^¶ To whom all correspondence should be addressed:
Kimmel Cancer Institute, BLSB 1002, 233 South 10th St., Philadelphia, PA 19107.
Tel.: 215-503-4650; Fax: 215-923-4498.

Abstract

Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-γ₁ to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P)⁹⁹²), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P)⁹⁹² receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (α-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P)⁹⁹² receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P)⁹⁹² receptors were associated with more SOS, Ras-GTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA-51093, CA-53149, and NS-31102 and a grant from the Ronald McDonald's Children's Charities. 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:

RPTK

receptor protein-tyrosine kinase

EGF

epidermal growth factor

EGFR

EGF receptor

Act. EGFR

activated EGF receptor

PLCγ₁

phospholipase C-γ₁

PLC-SH2

PLC-γ₁ SH2 domain fusion protein

Tyr(P)⁹⁹²

phosphorylated tyrosine 992 of the EGF receptor

GST-EGFR-(987-1028)

fusion protein of amino acids 987-1028 of the EGF receptor

GAP

Ras-GTPase-activating protein

SOS

Son of Sevenless protein

SHPTP2

SH2 domain containing phosphotyrosine phosphatase 2

Grb2

growth factor receptor-binding protein 2

PI

phosphatidylinositol

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

FITC

fluorescein isothiocyanate

GST

glutathione S-transferase.
↵² D. R. Emlet and A. J. Wong, unpublished observations.
- Received September 11, 1996.
- Revision received November 15, 1996.