Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.

Abstract

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4~ and processed for transmission electron microscopy. Under these conditions, ~ 6 • 105 molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37~ cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37~ 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing I~I-EGF and fluorescein-conjugated EGF.KEY WORDS hormone receptors growth factor 9 endocytosis lysosomes Epidermal growth factor (EGF) ~ initiates a complex series of cellular events which ultimately 1 Abbreviations used in this paper: DMEM/BSA, Dulbecco's Modified Eagle Medium containing 0.1% bovine serum albumin; EGF, epidermal growth factor; I~I-EGF, l~ZI-labeled epidermal growth factor; MVB, multivesicular body; PBS, 9.5 mM sodium phosphate (pH 7.4) containing 4.14 mM KCI and 0.137 M NaCI.results in increased DNA synthesis and cell division (see references 6 and 7 for reviews). Cultured cells that are responsive to EGF contain specific, saturable plasma membrane receptors for this hormone (9, 19). As a first step in examining the biochemical mechanism by which EGF exerts its growth-promoting effects, we have investigated the metabolic fate of EGF using both ~I-labeled EGF and fluorescein-conjugated EGF. Our studies (4, 17) have indicated that cell-bound EGF rapidly is internalized into endocytic vesicles and 382J. CELL BIOLOGY 9 The Rockefeller University Press.

Regulation of PTEN/Akt and MAP kinase signaling pathways by the ubiquitin ligase activators Ndfip1 and Ndfip2

“…S1). This suggests that the EGFR meets the Ndfip proteins following its endocytosis, which rapidly follows EGF binding (20).…”

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…It is worth noting that the binding of EGF ligand to its cognate receptor at ambient temperature (25°C ≤ temp ≤ 37°C), initiates a signaling cascade that involves the internalization of the activated receptor into endocytic vesicles of the cell (49,50). Figure 4D shows the confocal microscopy image of a QD585-M2 bead (160,000 Qdots/bead) next to a Qdot-EGF ligand activated A431 cell (100,000 Qdots per cell) (51).…”

When cell biology meets development: endocytic regulation of signaling pathways

“…A role for endocytosis in the regulation of RTK signaling was first proposed in the late 1970s when it was observed that the EGFR was internalized following application of ligand (Haigler et al 1979). Since the mid-1980s, the role that endocytosis plays in regulating EGFR signaling has been the subject of intense investigation and controversy (Leof 2000;Ceresa et al 2001).…”