Oxidative iodine monochloride iodination technique

Doran, D. M.; Spar, Irving L.

doi:10.1016/0022-1759(80)90304-x

Cited by 39 publications

(16 citation statements)

References 15 publications

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“…125 I-EGF Synthesis and Binding-125 I-EGF was synthesized using the oxidative ICl procedure of Doran and Spar (28). Radioligand binding was performed as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

Adak

DeAndrade

Pike

2011

Journal of Biological Chemistry

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The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-toback dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use 125 I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys 558 -Cys 567 disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys 571 -Cys 593 disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys 571 -Cys 593 disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunitsubunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.The EGF receptor is a receptor-tyrosine kinase composed of an extracellular ligand binding domain, a single ␣-helical transmembrane domain, and a cytoplasmic tyrosine kinase domain (1). In the absence of ligand, the EGF receptor exists as a monomer, although substantial evidence suggests that it is also present as an inactive predimer (2-5). Binding of EGF induces dimerization of the receptor and leads to the stimulation of its intracellular tyrosine kinase activity (6).The primary target of this tyrosine kinase is the receptor itself, with phosphorylation occurring in trans on the C-terminal tail of the receptor (7). These phosphotyrosine residues serve as sites for the binding of Src homology 2 and PTB domain-containing proteins that promote the assembly of the signaling complexes that mediate the intracellular effects of EGF (8 -11).The extracellular domain of the EGF receptor is composed of four subdomains referred to as subdomains I-IV (12, 13). Subdomains I and III are homologous and together form the site at which EGF is bound. Subdomains II and IV are also homologous and are regions of high cysteine content. In the absence of ligand, the extracellular domain is held in a closed conformation by interactions between the dimerization arm in subdomain II and the tethering arm in subdomain IV (14). Upon b...

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“…125 I-EGF Synthesis and Binding-125 I-EGF was synthesized using the oxidative ICl procedure of Doran and Spar (28). Radioligand binding was performed as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

Adak

DeAndrade

Pike

2011

Journal of Biological Chemistry

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“…Polyhalo astatine ions AtX 2 (X = Cl, Br, I) were also used recently to stabilize positive astatine [189]. *IC1 is most easily obtained by equilibration of radioactive iodide with ICI and has been used since 1958 for protein labeling [190,191]. It is still used for this purpose [192,193], although only low specific activities can be obtained.…”

Section: Direct Electrophilic Halogenationmentioning

confidence: 99%

No-Carrier-Added Radiohalogenation Methods with Heavy Halogens

1983

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Radiolabeling methods with the heavy halogens bromine, iodine and astatine are reviewed. The methods are evaluated on the basis of their underlying chemical principles and their usefulness for the no-carrier-added labeling of biologically-useful compounds. Special emphasis is placed on the stability, specific activity, and quality control of radiohalogenation products using the shortlived, neutron-deficient nuclides 7s ' 7 'Br, '"I, and 2 "At in view of their importance as radiopharmaceuticals.

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“…125 I-EGF was synthesized using the ICl method of Doran and Spar (24). The cells were plated into 6-well dishes 48 h prior to assay and grown in DMEM supplemented with 10% Fetalplex (Gemini Bio-Products, West Sacramento, CA), penicillin/streptomycin, and the desired concentration of doxycycline.…”

Section: Dna Constructs and Cell Lines-thementioning

confidence: 99%

The Membrane-proximal Intracellular Domain of the Epidermal Growth Factor Receptor Underlies Negative Cooperativity in Ligand Binding

Adak

Yang²,

Macdonald-Obermann

et al. 2011

Journal of Biological Chemistry

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Background:The EGF receptor exhibits negative cooperativity in ligand binding. Results: Mutation of the most membrane-proximal portion of the intracellular domain abrogates negative cooperativity. Conclusion: Residues 645-665 of the EGF receptor are involved in the genesis of negative cooperativity.Significance: These data demonstrate how alterations of the intracellular domain of the EGF receptor can lead to changes in ligand binding by the extracellular domain.

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Oxidative iodine monochloride iodination technique

Cited by 39 publications

References 15 publications

The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

No-Carrier-Added Radiohalogenation Methods with Heavy Halogens

The Membrane-proximal Intracellular Domain of the Epidermal Growth Factor Receptor Underlies Negative Cooperativity in Ligand Binding

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