Single molecule imaging of the signaling activity of the epidermal growth factor receptor
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Date
2014-02-25
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Abstract
Der EGF-Rezeptor (engl. epidermal growth factor), auch ErbB1 genannt, gehört zu den Rezeptor-Tyrosinkinasen und ist an verschiedenen zellulären Prozessen, wie der Proliferation und der Differenzierung, beteiligt ist. Die extrazellulären Domänen des Rezeptors können verschiedene Liganden binden, unter anderem EGF. Teile dieser extrazellulären Domänen, sowie die Transmembrandomäne und die Juxtamembran-Domäne spielen eine Rolle bei der Dimerisierung des Rezeptors. Nach Bindung eines Liganden bildet die intrazelluläre Kinasedomäne ein asymmetrisches Dimer mit der Kinase eines zweiten Rezeptors, wodurch die Autophosphorylierung verschiedener Serine und Tyrosine am C-terminalen Ende des Rezeptors stattfindet. Dort binden Effektorproteine, welche das Signal in die Zelle weiterleiten. In dieser Arbeit konnte der kurzlebige Charakter der Rezeptor-Dimerisierung und der Cluster- Bildung durch die Verfolgung fluoreszenzmarkierter Rezeptoren in lebenden Zellen auf Einzelmolekül-Ebene visualisiert werden. Gleichzeitig wurde die Aktivität des Rezeptors durch eine Sonde für den Phosphorylierungszustand verfolgt. Auch nach Aktivierung wechselt ErbB1 permanent zwischen mehreren kurzlebigen Mobilitätszuständen, deren Diffusionskoeffizienten einen Bereich von zwei Größenordnungen umfassen. Dies verdeutlicht, dass die Aktivierung von ErbB1 ein hochdynamischer Prozess ist. In unstimulierten Zellen liegt der Rezeptor hauptsächlich monomer vor, was gegen eine entscheidende Rolle der Vordimerisierung bei der Aktivierung von ErbB1 spricht. Nach Stimulierung mit EGF assoziieren die Rezeptoren und lokalisieren in hochaktiven, immobilen Clustern, welche mit Clathrinumhüllten Einstülpungen kolokalisieren. Demnach wird das Signal durch die Rekrutierung der Rezeptoren zu Clathrin-umhüllten Einstülpungen vor der Endozytose an der Membran stabilisiert und amplifiziert. Allerdings sind die Rezeptoren dort nicht unbedingt gefangen, da sie immer noch zwischen den Mobilitätszuständen wechseln können. Experimente mit einem Kinaseinhibitor und einer Kinase-inaktiven Mutante des Rezeptors zeigten, dass diese zwar den immobilen Zustand erreichen, aber dass für die Cluster-Bildung auch die Kinaseaktivität benötigt wird. Zudem zeigten Experimente mit einem Phosphataseinhibitor, dass die Bindung eines Liganden für die Cluster-Bildung aktiver ErbB1 Rezeptoren nicht notwendig ist. In Zellen mit geringem Expressionslevel von ErbB1, ist die meiste Aktivität des Rezeptors räumlich beschränkt in Clathrin-umhüllten Einstülpungen zu finden, wohingegen eine gleichmäßige Verteilung der Aktivität bei Überexpression beobachtet wird. Die Anhäufung in hochaktiven Membranbereichen beschreibt einen möglichen Mechanismus, um bei geringem Expressions-level des Rezeptors eine stabile Signalweiterleitung zu gewährleisten.
SUMMARY The epidermal growth factor receptor (ErbB1) is a receptor tyrosine kinase involved in various cellular processes, such as growth and differentiation. Its extracellular domains are capable of binding a range of ligands, including the epidermal growth factor (EGF). Parts of these extracellular domains, as well as the transmembrane domain and the juxtamembrane domain are involved in dimerization of the receptor. Upon ligand binding, the intracellular kinase domain forms an asymmetric dimer with the kinase of a second receptor, leading to autophosphorylation of several tyrosine and serine residues at the C-terminal tail, where effector proteins bind to propagate the signal. In this work the transient nature of receptor dimerization and clustering was visualized by tracking a fluorescently labeled receptor in living cells on a single molecule level. At the same time the activity of the receptor was monitored with a probe for phosphorylation. Even after activation, ErbB1 continuously switches between multiple short-lived mobility states, with diffusion coefficients that span two orders of magnitude, indicating that ErbB1 activation is a highly dynamic process. In resting cells the receptor is mainly monomeric arguing against a role of pre-dimerization in the activation of ErbB1. Upon stimulation with EGF, the receptor self-associates and localizes in highly active, immobile clusters that colocalize with clathrincoated pits. Thus, ErbB1 signaling is stabilized and amplified at the plasma membrane by recruitment of the receptors to clathrin-coated pits prior to endocytosis. However, the receptors are not necessarily trapped, as they maintain their ability to alternate between mobility states. Experiments with a kinase inhibitor and a kinase-dead mutant of ErbB1 showed that although receptors with an inactive kinase are able to convert to the immobile state, kinase activity is necessary for cluster formation. Experiments with a phosphatase inhibitor showed that ligand binding is not necessary for clustering of active ErbB1. In cells expressing the receptor at a low level, much of the ErbB1 activity is spatially confined to clathrin-coated pits on the plasma membrane, in contrast to the uniform activity observed in over-expressing cells. This clustering into highly active areas represents a potential mechanism for robust signaling from the plasma membrane at low expression levels of the receptor.
SUMMARY The epidermal growth factor receptor (ErbB1) is a receptor tyrosine kinase involved in various cellular processes, such as growth and differentiation. Its extracellular domains are capable of binding a range of ligands, including the epidermal growth factor (EGF). Parts of these extracellular domains, as well as the transmembrane domain and the juxtamembrane domain are involved in dimerization of the receptor. Upon ligand binding, the intracellular kinase domain forms an asymmetric dimer with the kinase of a second receptor, leading to autophosphorylation of several tyrosine and serine residues at the C-terminal tail, where effector proteins bind to propagate the signal. In this work the transient nature of receptor dimerization and clustering was visualized by tracking a fluorescently labeled receptor in living cells on a single molecule level. At the same time the activity of the receptor was monitored with a probe for phosphorylation. Even after activation, ErbB1 continuously switches between multiple short-lived mobility states, with diffusion coefficients that span two orders of magnitude, indicating that ErbB1 activation is a highly dynamic process. In resting cells the receptor is mainly monomeric arguing against a role of pre-dimerization in the activation of ErbB1. Upon stimulation with EGF, the receptor self-associates and localizes in highly active, immobile clusters that colocalize with clathrincoated pits. Thus, ErbB1 signaling is stabilized and amplified at the plasma membrane by recruitment of the receptors to clathrin-coated pits prior to endocytosis. However, the receptors are not necessarily trapped, as they maintain their ability to alternate between mobility states. Experiments with a kinase inhibitor and a kinase-dead mutant of ErbB1 showed that although receptors with an inactive kinase are able to convert to the immobile state, kinase activity is necessary for cluster formation. Experiments with a phosphatase inhibitor showed that ligand binding is not necessary for clustering of active ErbB1. In cells expressing the receptor at a low level, much of the ErbB1 activity is spatially confined to clathrin-coated pits on the plasma membrane, in contrast to the uniform activity observed in over-expressing cells. This clustering into highly active areas represents a potential mechanism for robust signaling from the plasma membrane at low expression levels of the receptor.
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Keywords
EGFR, Fluoreszenzmikroskopie, Single particle tracking