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Conformational coupling across the plasma membrane in activation of the EGF receptor.

ABSTRACT: 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. Although 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.

SUBMITTER: Endres NF

PROVIDER: S-EPMC3718647 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Conformational coupling across the plasma membrane in activation of the EGF receptor.

Endres Nicholas F NF Das Rahul R Smith Adam W AW Arkhipov Anton A Kovacs Erika E Huang Yongjian Y Pelton Jeffrey G JG Shan Yibing Y Shaw David E DE Wemmer David E DE Groves Jay T JT Kuriyan John J

Cell 20130101 3

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. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular modu ...[more]

PMID: 23374349

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Project description:CLC anion channels are homodimeric proteins. Each subunit is comprised of 18 α-helices designated "A-R" and an intracellular carboxy-terminus containing two cystathionine-β-synthase (CBS1 and CBS2) domains. Conformational coupling between membrane and intracellular domains via poorly understood mechanisms is required for CLC regulation. The activity of the C. elegans CLC channel CLH-3b is reduced by phosphorylation of a carboxy-terminus "activation domain," which disrupts its interaction with CBS domains. CBS2 interfaces with a short intracellular loop, the H-I loop, connecting membrane helices H and I. Alanine mutation of a conserved H-I loop tyrosine residue, Y232, prevents regulation demonstrating that the loop functions to couple phosphorylation-dependent CBS domain conformational changes to channel membrane domains. To gain further insight into the mechanisms of this coupling, we mutated conserved amino acid residues in membrane helices H and I. Only mutation of the H-helix valine residue V228 to leucine prevented phosphorylation-dependent channel regulation. Structural and functional studies of other CLC proteins suggest that V228 may interact with Y529, a conserved R-helix tyrosine residue that forms part of the CLC ion conduction pathway. Mutation of Y529 to alanine also prevented CLH-3b regulation. Intracellular application of the sulfhydryl reactive reagent MTSET using CLH-3b channels engineered with single-cysteine residues in CBS2 indicate that V228L, Y529A, and Y232A disrupt putative regulatory intracellular conformational changes. Extracellular Zn2+ inhibits CLH-3b and alters the effects of intracellular MTSET on channel activity. The effects of Zn2+ are disrupted by V228L, Y529A, and Y232A. Collectively, our findings indicate that there is conformational coupling between CBS domains and the H and R membrane helices mediated by the H-I loop. We propose a simple model by which conformational changes in H and R helices mediate CLH-3b regulation by activation domain phosphorylation.

Dataset Information

Conformational coupling across the plasma membrane in activation of the EGF receptor.

Publications

Conformational coupling across the plasma membrane in activation of the EGF receptor.

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