Cytoskeletal association of epidermal growth factor receptor and associated signaling proteins is regulated by cell density in IEC‐6 intestinal cells

Epidermal growth factor (EGF) mediates a variety of physiologic responses in rat intestine. EGF receptor (EGFR) responsiveness to EGF is mediated by the surface expression of high affinity EGFR, which is associated with the cytoskeleton (CSK). EGFR signal transduction appears to be mediated by the CSK association of EGFR and related signaling proteins. In the nontransformed intestinal cell line IEC‐6, expression of EGFR, Src homology and collagen protein (SHC), phospholipase Cγ1 (PLCγ), and their tyrosine phosphorylation in response to EGF was assayed by immunoblot. The distribution of EGFR and tyrosine‐phosphorylated EGFR was regulated by cell density. At confluence, EGFR and tyrosine‐phosphorylated EGFR were predominantly associated with the Triton X‐100‐insoluble CSK at confluence, while predominantly Triton X‐100‐soluble at subconfluence. PLCγ was predominantly soluble at both states of confluence. Confluent but not subconfluent IEC‐6 cells demonstrated a cascade of EGF‐mediated events consisting of a transient CSK association of PLCγ with EGFR, a brief expression of tyrosine‐phosphorylated PLCγ, a brief increase in PLCγ CSK association, and a prolonged soluble association of PLCγ with the EGFR. EGF led to an increase in the CSK association of SHC at both states of confluence and was greater at confluence. EGFR association with SHC was primarily soluble at subconfluence, while at confluence EGFR association was markedly increased and predominantly in the CSK. Thus, cell density regulates the CSK association of the EGFR and its ability to associate and activate signaling pathways in intestinal cells. J. Cell. Physiol. 172:126–136, 1997. © 1997 Wiley‐Liss, Inc.