The Human Epidermal Growth Factor Receptor Contains a Juxtamembrane Calmodulin-Binding Site

Martı́n-Nieto, José; Villalobo, Antonio

doi:10.1021/bi971765v

Cited by 105 publications

(141 citation statements)

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“…The standard distances of 1.70 -1.80, 2.20 -3.10, and 2.50 -2.70 Å were used to calibrate lower and upper bounds for geminal protons, vicinal protons, and proton vicinal to methyl, respectively. If neither were observed, the largest calibration factor found for the other 13 C was used. An extra 0.5 Å was added to the upper distance restraint for NOEs involving methyl groups.…”

Section: Methodsmentioning

confidence: 99%

“…Several intrinsic sorting signals and protein binding sites have now been mapped to the JX region, which regulates receptor trafficking and inactivation. These include basolateral sorting signals (10), a lysosomal sorting motif (11), a nuclear localization signal (12), as well as binding sites for calmodulin (13), ␣-subunits of heterotrimeric G s proteins (14), and phosphoinositide kinases (15). The JX region also includes post-translational modification sites; Thr 654 is a known substrate for PKC (16), and Thr 669 and Ser 671 are substrates for MAPK (17,18).…”

mentioning

confidence: 99%

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

Choowongkomon

Carlin

Sönnichsen

2005

Journal of Biological Chemistry

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The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxtamembrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking. Two sorting signals, a PXXP motif and a 658 LL 659 motif, are responsible for basolateral sorting in polarized epithelial cells, and a 679 LL 680 motif targets the ligand-activated receptor for lysosomal degradation. To understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in aqueous solution and in dodecylphosphocholine (DPC) detergent. JX is inherently unstructured in aqueous solution, albeit a nascent helix encompasses the lysosomal sorting signal. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. A large, internally inconsistent group of long range nuclear Overhauser effects suggest a close proximity of the helices, and the presence of significant conformational averaging. Models were determined for the average JX conformation using restraints representing the translational restriction due to micelle-surface adsorption, and the helix orientations were determined from residual dipolar couplings. Two equivalent average structural models were obtained that differ only in the relative orientation between first and second helices. In these models, the 658 LL 659 and 679 LL 680 motifs are located in the first and second helices and face the micelle surface, whereas the PXXP motif is located in a flexible helix-connecting region. The data suggest that the activity of these signals may be regulated by their membrane association and restricted accessibility in the intact receptor.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Choowongkomon

Carlin

Sönnichsen

2005

Journal of Biological Chemistry

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“…RLKs are involved in mediating developmental and defense-related cellular responses by binding extracellular ligands and activating downstream signaling pathways (18). Moreover, interactions with CaM, phosphorylation, or dephosphorylation are processes shown to activate some animal RLKs (19). Arabidopsis Clavata 1, RLK4, SFR1, BRI1, and CRCK1 were shown to be CaM targets (20).…”

Section: Cam Interaction With Receptor-like Protein Kinases (Rlks)mentioning

confidence: 99%

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Popescu

Popescu²,

Bachan³

et al. 2007

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

345

292

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Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/ CML proteins to regulate a wide range of targets and cellular activities.calmodulin-interacting proteins ͉ protein interaction network

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“…Although one must obviously be cautious when interpreting cell biological experiments with CaM inhibitors, we do wish to suggest that they can provide useful information about cellular processes. For example, Ca 2ϩ /CaM binds to the EGFR (18,20) and to peptides corresponding to its JM region (19,21,53). Does this binding contribute to the activation of the receptor?…”

Section: W-7/w-13/w-12 Bind Strongly To Phospholipid Membranes Reducmentioning

confidence: 99%

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

Sengupta

Ruano

Tebar

et al. 2007

Journal of Biological Chemistry

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Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca 2؉ /CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca 2؉ /CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factordependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca 2؉

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The Human Epidermal Growth Factor Receptor Contains a Juxtamembrane Calmodulin-Binding Site

Cited by 105 publications

References 50 publications

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

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

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

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

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