Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells

D. A. Jans, L. Bergmann, R. Peters, F. Fahrenholz

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Abstract

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2,4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8]vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with uorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.

Original languageEnglish
Pages (from-to)14599-14605
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number24
Publication statusPublished - 10 Sep 1990
Externally publishedYes

Cite this

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title = "Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells",
abstract = "Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2,4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8]vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with uorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.",
author = "Jans, {D. A.} and L. Bergmann and R. Peters and F. Fahrenholz",
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Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells. / Jans, D. A.; Bergmann, L.; Peters, R.; Fahrenholz, F.

In: Journal of Biological Chemistry, Vol. 265, No. 24, 10.09.1990, p. 14599-14605.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells

AU - Jans, D. A.

AU - Bergmann, L.

AU - Peters, R.

AU - Fahrenholz, F.

PY - 1990/9/10

Y1 - 1990/9/10

N2 - Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2,4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8]vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with uorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.

AB - Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2,4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8]vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with uorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.

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