Subsets of epidermal growth factor receptors during activation and endocytosis.
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-gamma1 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 (alpha-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.
Emlet
D R
DR
Department of Pharmacology, Kimmel Cancer Institute, Philadelphia, Pennsylvania 19107, USA.
Moscatello
D K
DK
Ludlow
L B
LB
Wong
A J
AJ
D000185:Actinin / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D004705:Endocytosis; D066246:ErbB Receptors / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism; D018909:src Homology Domains
D000185:Actinin; D066246:ErbB Receptors; D010738:Type C Phospholipases