Volume 272, Number 7, Issue of February 14, 1997 pp. 4079-4086
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Subsets of Epidermal Growth Factor Receptors during Activation and Endocytosis

(Received for publication, September 11, 1996, and in revised form, November 15, 1996)

David R. Emlet , David K. Moscatello ^§ , Laural B. Ludlow ^§ and Albert J. Wong ^§

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

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-₁ 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 (-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.

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