A Structural Model for the Membrane-bound Form of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

Choowongkomon, Kiattawee; Carlin, C.; Sönnichsen, Frank D.

doi:10.1074/jbc.m502698200

Cited by 42 publications

(41 citation statements)

References 67 publications

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“…In agreement with the crystal structure, the region just before Gly672 (i.e., Glu661-Gly672), which is negatively charged and contains a central PxxP sequence, is unstructured (48). Our results appear to disagree with the solution NMR studies: the N-terminal JM helix (residues 652-661) observed in their studies overlaps the unstructured JM sequence in our TM-JM peptides.…”

Section: Discussionsupporting

confidence: 71%

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin

et al. 2006

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The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.

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Section: Discussionsupporting

confidence: 71%

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin

et al. 2006

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“…Molecular dynamics simulations suggest that when EGFR is in an inactive conformation, the LRRLL motif within the JM-A segment is buried in the membrane (Arkhipov et al), consistent with NMR data for the isolated juxtamembrane segment of EGFR in detergent micelles (Choowongkomon et al, 2005). These observations suggest that the nature of the interaction between the transmembrane helices toggles the configuration and membrane association of the JM-A portion of the juxtamembrane segments (Figure 7A).…”

Section: Discussionsupporting

confidence: 70%

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

Endres

Das

Smith

et al. 2013

Cell

439

632

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Summary How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. While the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

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“…Upon N-terminal TMD dimerization, the JM-A segments interact with the membrane and form an α-helical, antiparallel homo-dimer (8,39,40). The α-helical structure of the JM-A dimer has been confirmed in NMR and MD simulation studies of TMD-JM-A fragments (2,8,41) and in MD simulation of the active EGFR dimer (8).…”

Section: Resultsmentioning

confidence: 70%

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Kaszuba

Grzybek

Orłowski

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

143

156

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The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.R eceptor tyrosine kinases (RTKs) are cell surface receptors that receive and transduce signals mediating a variety of critical cellular processes, including cell growth, migration, proliferation, differentiation, and apoptosis. Among the many RTKs, the most studied is the epidermal growth factor receptor (EGFR), not least because of its involvement in the development and progression of epidermoid cancers and its resulting importance as a target for antineoplastic therapies.Structurally, the EGFR consists of the ectodomain (ECD) (further subdivided into four subdomains, DI-IV), the transmembrane domain (TMD), and the intracellular tyrosine kinase domain (TKD). Ligand binding induces conformational transitions of the ECD that stabilize receptor dimerization, culminating in the activation of the intracellular TKD and subsequent propagation of the activation signal (1). To prevent receptor activation and signaling in the absence of ligand, the structurally tethered ECD of monomeric EGFR blocks the intrinsic capacity of the TMD and the intracellular TKD to dimerize (2). Ligand binding is believed to release the self-inhibitory tether and facilitate receptor oligomerization and activation (3-6). A detailed understanding of the structural regulation of the intact full-length receptors in their native membranes promises to reveal the molecular basis for receptor regulation (7); however, the methodological limitations associated with crystallizing transmembrane proteins, together with the high flexibility of the full-length receptor, have prevented high-resolution crystallographic analysis.To fill this gap, extensive atom-scale molecular dynamics (MD) simulations were recently performed to elucidate the structural dynamics of the EGFR in a two-component lipid bilayer (8). These studies suggest a large interfacial contact area between the membrane and the ecto-and intracellular domains of the unli...

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A Structural Model for the Membrane-bound Form of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

Cited by 42 publications

References 67 publications

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

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A Structural Model for the Membrane-bound Form of the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

Cited by 42 publications

References 67 publications

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca2+/Calmodulin

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca2+/Calmodulin

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

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Product

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Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca²⁺/Calmodulin