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

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.