Reduced affinity of lodinated forms of Tyr0 C-type natriuretic peptide for rat natriuretic peptide receptor B

Gaétan Thibault, Kevin Grove, Christian F. Deschepper

Research output: Contribution to journalArticle

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Abstract

Tyr0CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr0CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr0CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always 0CNP, most likely by changing thethiolof the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr0CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e., Tyr0Met(O)17CNP, 127I-Tyr°Met(O)17CNP, and 127I-Tyr0Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr0CNP, ANP(99-126), BNP-32, and des[Gln18,Ser19,Gly20,Leu21,Gly 22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr0CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr0CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr0CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.

Original languageEnglish (US)
Pages (from-to)1046-1053
Number of pages8
JournalMolecular Pharmacology
Volume48
Issue number6
StatePublished - Dec 1995
Externally publishedYes

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C-Type Natriuretic Peptide
Atrial Natriuretic Factor
sulfoxide
Halogenation
COS Cells
Iodine
Ligands
Natriuretic Peptides
Peptides
Membranes
Brain Natriuretic Peptide
Tyrosine
atrial natriuretic factor receptor B
Plasmids
High Pressure Liquid Chromatography
atrial natriuretic factor receptor A

ASJC Scopus subject areas

  • Pharmacology

Cite this

Reduced affinity of lodinated forms of Tyr0 C-type natriuretic peptide for rat natriuretic peptide receptor B. / Thibault, Gaétan; Grove, Kevin; Deschepper, Christian F.

In: Molecular Pharmacology, Vol. 48, No. 6, 12.1995, p. 1046-1053.

Research output: Contribution to journalArticle

Thibault, Gaétan ; Grove, Kevin ; Deschepper, Christian F. / Reduced affinity of lodinated forms of Tyr0 C-type natriuretic peptide for rat natriuretic peptide receptor B. In: Molecular Pharmacology. 1995 ; Vol. 48, No. 6. pp. 1046-1053.
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abstract = "Tyr0CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr0CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr0CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always 0CNP, most likely by changing thethiolof the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr0CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e., Tyr0Met(O)17CNP, 127I-Tyr°Met(O)17CNP, and 127I-Tyr0Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr0CNP, ANP(99-126), BNP-32, and des[Gln18,Ser19,Gly20,Leu21,Gly 22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr0CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr0CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr0CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.",
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AU - Deschepper, Christian F.

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N2 - Tyr0CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr0CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr0CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always 0CNP, most likely by changing thethiolof the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr0CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e., Tyr0Met(O)17CNP, 127I-Tyr°Met(O)17CNP, and 127I-Tyr0Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr0CNP, ANP(99-126), BNP-32, and des[Gln18,Ser19,Gly20,Leu21,Gly 22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr0CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr0CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr0CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.

AB - Tyr0CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr0CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr0CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always 0CNP, most likely by changing thethiolof the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr0CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e., Tyr0Met(O)17CNP, 127I-Tyr°Met(O)17CNP, and 127I-Tyr0Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr0CNP, ANP(99-126), BNP-32, and des[Gln18,Ser19,Gly20,Leu21,Gly 22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr0CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr0CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr0CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.

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