Regulation of EGFR by the NF2 tumor suppressor, Merlin

Neurofibromatosis type 2 (NF2) is a familial tumor syndrome in which patients exhibit heterozygosity at the NF2 locus and develop multiple benign tumors subsequent to somatic inactivation of the remaining wild-type allele. Analyses of non-NF2 tumor samples also reveal homozygous NF2 mutations in some spontaneous cancers, including renal cell carcinoma (RCC). In vitro inactivation of NF2 characteristically results in loss of contact-dependent inhibition of proliferation. However, in many cell types this can be rescued by inhibition of epidermal growth factor receptor (EGFR) signaling. To evaluate the significance of the regulation of EGFR by NF2 in vivo, I targeted inactivation of murine Nf2 in cells of the renal tubule epithelia. All of these mice developed renal carcinomas and cells in these tumors required EGFR signaling for proliferation. This valuable mouse model supports a role for EGFR in tumorigenesis from NF2-deficient cells in vivo and gives added credence to the roles of EGFR and perhaps NF2 in RCC.Tumor suppressor activity of the NF2 gene product, Merlin, requires its localization to the cortical cytoskeleton. Like the closely related ERM proteins, Merlin is thought to tether this mesh-like structure to certain proteins and phospholipids in the plasma membrane. Merlin physically associates with EGFR and prevents its ligand-induced internalization and signaling in a contact-dependent manner. Using single particle tracking microscopy and Texas-red-conjugated EGF to monitor lateral mobility and internalization of EGFR, respectively, we show that ligand-bound EGFR is immobilized in the plasma membrane of confluent Merlin-expressing cells in a contact-, actin-, and signaling-dependent manner. Ligand-activated EGFR undergoes receptor-mediated internalization via either clathrin-mediated endocytosis (CME) or non-clathrin endocytosis (NCE). Surprisingly, we found that Merlin specifically antagonizes NCE of EGFR independent of cell-cell contact by preventing distribution of the receptor to specific membrane microdomains. These contact-dependent and -independent functions of Merlin are both necessary for appropriate regulation of EGFR internalization. The transition between these roles is mediated by phosphorylation-induced conformation changes in the head-to-tail association of Merlin. These studies illuminate a novel mechanism of contact-dependent feedback inhibition of EGFR and provide insight on the molecular function of Merlin and the regulation of EGFR internalization.