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

Abstract

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.

Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential

“…As expected from the model, sphingosine activates EGFR in the absence of EGF (85) at the same concentration (2 M) at which it binds to membranes and reverses the sign of the zeta potential (21). Furthermore, fluorescence experiments show that sphingosine causes the JM region of reconstituted transmembrane ϩ JM EGFR peptides to desorb from bilayer membranes (25).…”

“…Recent experiments support the claim that the JM region can bind with high affinity to the inner leaflet of the plasma membrane. Specifically, NMR and fluorescence measurements on peptides corresponding to transmembrane plus JM domains of EGFR reconstituted into phospholipid vesicles (25) show the transmembrane helix breaks at the membranesolution interface, and the extended JM region binds to the membrane if it contains acidic lipids (either 1% phosphatidylinositol 4,5-bisphosphate or Ͼ5% PS); adding Ca 2ϩ /CaM can release the JM region from the membrane.…”

“…A recent report provides important evidence that ligand-mediated activation of EGFR involves dimer formation between two kinase domains, which stimulates rearrangement of the activation loop within the kinase domain by an allosteric mechanism (22)(23)(24). Ca 2ϩ / CaM, for example, could facilitate formation of the kinase domain dimers by enhancing desorption of the JM plus kinase domains from the cytoplasmic leaflet (7,25).…”

Receptor tyrosine kinase transmembrane domains

“…Amino acids in this region have been shown to serve as binding and phosphorylation sites for signaling molecules. [45][46][47][48][49][50] The importance of the JM domain in signaling is evident from several mutations, deletions and insertions in this domain that can lead to cancer. [51][52][53] In addition, recent studies have provided evidence for the active role of the JM domains in regulating RTK activity.…”

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

“…Different mechanistic models have been proposed to account for the stimulatory action of Ca 2 + -CaM on EGFR activation [27]. The most likely mechanism involves the Ca 2 + -CaM-induced release of the positively-charged CaM-BD from the phosphoinositiderich negatively-charged inner leaflet of the plasma membrane, an electrostatic interaction which otherwise would maintain the ligand-free receptor auto-inhibited [22,25,28]. Most significantly, the intracellular juxtamembrane region of the receptor, which contains the CaM-BD, has been shown to be indispensable for the allosteric activation mechanism mentioned above to be operative Abbreviations: CaM, calmodulin; CaM-BD, CaM-binding domain; c-Src, cellular sarcoma kinase; DMEM, Dulbecco's modified Eagle's medium; DSS, disuccinimidyl suberate; EGFR, epidermal growth factor receptor; ErbB, avian erythroblastosis oncogene B homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; NOS, nitric oxide synthase; P-(Tyr)-CaM, tyrosine-phosphorylated CaM; PDE1, phosphodiesterase 1; P-EGFR, auto(trans)-phosphorylated receptor; PGT, poly-L-(Glu:Tyr).…”