Recruitment of Epidermal Growth Factor Receptors into Coated Pits Requires Their Activated Tyrosine Kinase

EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligandinduced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s). T HE binding of EGF to its cell surface receptor triggers intracellular signaling pathways through a welldefined sequence of events to regulate cell proliferation and development (Cantley et al., 1991). Upon ligand binding, EGF-receptors (EGF-R) ~ dimerize and the intrinsic tyrosine kinase becomes activated leading to autophosphorylation of the receptor. Specific intracellular substrates involved in the metabolic and mitogenic responses to EGF are recruited to the newly created phosphotyrosine-containing binding sites on the EGF-R via their SH2 domains (Carpenter, 1992). EGF binding also triggers efficient receptor internalization. Internalized receptors are segregated from the default recycling pathway and are routed to lysosomes where they are degraded. The resulting decrease in the number of receptors expressed at the cell surface is termed receptor down-regulation (Haigler et al., 1979). Down-regulation of activated EGF-R is believed to play an essential role in modulating the cell's proliferative response to the growth hormone (Wells et al., 1990). Mutation of the intrinsic tyrosine kinase activity of the EGF-R abolishes both signaling for biological responses and ligand-induced down-regulation (Chen et al., 1987; Honneger et al., 1987). The question of whether the tyrosine kinase of the EGF-R is directly required for its internalization Address all correspondence to Sandra L. Schmid, Department of Cell Biology, The Scripps Research Institute, La Jolla, California, 92037. Tel.: (619) 554-2311. Fax: (619)-554-6253. 1. Abbreviations used in this paper: B-EGE biotinylated-EGF; B-Tfn, biotinylated-Tfn; EGF-R, EGF receptor; Tfn, transferrin; wt, wild type. remains disputed (Sorkin and Waters, 1993). Comparable studies on EGF internalization in transformed cells expressing wild-type or kinase-deficient mutant EGF-R have led to contradictory conclusions. Some studies reported that kinase-deficient receptors are not efficiently internalized (Glenney et al., 1988; Chen et al., 1989; Wiley et al., 1991). Others argue that kinase-deficient receptors are internalized as efficiently as wild-type receptors in response to EGF (Honneger et al., 1987; Felder et al., 1992) and instead suggest that the EGF-R tyrosine kinase activity prevents receptor recycling and is required to target internalized EGF-R to lysosomes for degradation (Felder et al., 1990; Honneger et al., 1990). One contributing factor in this debate stems from the difficulty in directly measuring the relative contributions of the rates of EGF-R endocytosis and recycling to the overall initial rate of accumulation of EGF in intact cells. To clarify this controversial issue, we studied the internalization of EGF in a novel cell-free assay that specifically and directly measures only the early events of endocytosis induced by EGF. This system uses perforated cells that are prepared by mechanical disruption so as to leave large gaps in their otherwise intact plasma membrane (Smythe et al., 1992). Cytosolic contents can then be removed allowing full access to exogenously added reagents. The recruitment of receptor-bound biotinylated EGF (B-EGF) into deeply invaginated, constricted coated pits is detected as it becomes inaccessible to exogenously added avidin (Lamaze et al., 1993). We have previously shown by both biochemical and morphological criteria, that the sequestration of B-EGF into constricted coated pits requires cytosolic factors that are distinct from those required for the efficient sequestration of the © The Rockefeller University Press, 0021-9525/95/0414718 $2.00 The Journal of Cell Biology, Volume 129, Number 1, April 1995 47-54 47 on A uust 4, 2017 jcb.rress.org D ow nladed fom constitutively internalized transferrin receptor. In addition, the sequestration of EGF but not transferrin (Tfn) is inhibited by the tyrosine kinase inhibitor, genistein. Using this assay we show that kinase-deficient receptors fail to undergo ligand-induced sequestration into coated pits. Sequestration of truncated EGF-R lacking the regulatory domain and its autophosphorylation sites also requires an active EGF-R tyrosine kinase. These results suggest that a kinase substrate(s), in addition to the EGF-R itself, is required for ligand-induced recruitment of activated EGF-R into coated pits. Finally, efficient recruitment of kinase-deficient EGF-R can be restored in trans by the addition of a soluble constitutively active EGF-R kinase. These results provide the first biochemical evidence that an activated tyrosine kinase is essential for the efficient ligand-induced recruitment of EGF-R into coated pits. Materials and Methods