Hormone receptor topology and dynamics: Morphological analysis using ferritin-labeled epidermal growth factor

Abstract

Previous studies using a biologically active 1:1 conjugate of EGF and ferritin (F-EGF) have traced the binding and internalization of the hormone molecules. In the present report, we develop ultrastructural criteria for identification of the F-EGF-receptor complex, and, thereby, enable utilization of the F-EGF as an indirect marker to localize the receptor for this peptide hormone. The ferritin cores of bound F-EGF are situated 4-6 nm from the extracellular surface of the membrane. When cells were incubated for up to 30 min at 370C, this characteristic spatial relationship was observed in all uptake stages (surface clustering, endocytosis, and incorporation into multivesicular bodies), indicating that the hormone receptor complex remains intact through these steps. However, when incubation was continued for periods sufficient to allow hormone degradation (30-60 min), pools of free ferritin were observed in lysosomes. In the presence of various amine inhibitors of hormone degradation, internalization and multivesicular body incorporation proceeded, but hormone'receptor degradation was blocked as evidenced by preservation of the ferritin-membrane relationship; i.e., no pools of free ferritin were seen after 60 min. These data provide morphological support for the hypothesis that down-regulation o surface receptors involves internalization of intact hormone-receptor complexes. In addition, we have developed a method for viewing the surface of intact cells en face, allowing closer scrutiny of the clustering of F-EGF' receptor complexes in the plane of the membrane prior to internalization. The particles in the F-EGF clusters observed by this method are spaced at 12 nm center-to-center, serving to set upper limits on the packing dimensions of the EGF-receptor complex. Although investigations into the mechanisms of hormone action have demonstrated cell surface binding and subsequent internalization of a number of polypeptide hormones-including insulin, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, and epidermal growth factor (EGF) (1-3)-a key question concerning the plasmalemmal hormone receptors remains unanswered: Are the receptors removed from the cell surface along with the hormone as part of the cellular response? Biochemical evidence that hormone binding capacity decreases after hormonal stimulation (down-regulation) suggests that receptors may be removed from the cell surface or modified to decrease binding capacity (4-7); however, the resolution of experimental techniques has been insufficient to differentiate clearly between these alternatives. Attempts to covalently link 125I-labeled EGF to its putative membrane receptor and follow its metabolic fate have been reported (8-10). The results suggest receptor internalization; however, only a small fraction of the membrane-bound EGF was successfully crosslinked, and many questions remain concerning the membrane sites, stability, and mode of down-regulation for the majority of EGF receptors.We recently have prepared an el...

Microtubules and microfilaments in fixed and permeabilized cells are selectively decorated by nerve growth factor.

“…This finding suggests that, if the pool of NGF internalized in target cells (3,5,9,18), presumably via endocytosis, is subsequently released and diffuses before being degraded, as is known to occur for some internalized proteins (24,25), the chances ofan interaction with these cytoskeletal structures are high.…”

Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane

“…However, only a few lipid-specifi c fl uorescent toxins are validated so far ( 45 ); and admittedly, their size is much greater than the targeted lipid (e.g., projected diameter of lysenin* is ‫ف‬ 15 times larger than endogenous SM). This size discrepancy predicts steric hindrance but does not preclude specifi city, as perhaps best exemplifi ed by EGFferritin conjugates for which grafting a ‫ف‬ 450 kDa ferritin moiety allowed us to faithfully follow the fate of the small EGF molecule [ ‫ف‬ 6 kDa; ( 46 )]. Complementarity between lipid analogs and lipid-specifi c toxins, when available, is illustrated by this paper.…”

Endocytosis and intracellular trafficking of ErbBs

“…2). EGF and EGFR begin to accumulate in the intralumenal membranes of MVB that are located in the perinuclear area of the cell after 15-20 min of EGF-induced endocytosis [80,81,88,89]. Serial sectioning electron microscopy demonstrated that internal membranes of MVB represent vesicles that are not connected to the limiting membrane [90].…”