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 fluorescein-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.
Jans
D A
DA
Max-Planck-Institut für Biophysik, Johann Wolfgang Goethe University, Frankfurt, Federal Republic of Germany.
Bergmann
L
L
Peters
R
R
Fahrenholz
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001127:Arginine Vasopressin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D051381:Rats; D011945:Receptors, Angiotensin / Q000032:analysis / Q000378:metabolism*; D017483:Receptors, Vasopressin; D013552:Swine
D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D001710:Biotin