Photoaffinity, biotinyl, and iodo analogs as probes for vasotocin receptors

Abstract

Vasotocin (AVT) analogues with tyrosine or phenylalanine in position 9, i.e., [9-tyrosine]AVT, [2-phenylalanine,-9-tyrosine]AVT, and 1-desamino[9-(p-aminophenylalanine)]AVT, were synthesized by the solid-phase method. These compounds showed a high biological activity in the hydroosmotic toad bladder assay. Using the chemically reactive functional groups on tyrosine and p-aminophenylalanine in position 9, we prepared iodinated, photoreactive, and affinity ligands, i.e., [2-phenylalanine,9-(iodotyrosine)]AVT, 1-desamino[9-(p-azidophenylalanine)]AVT, and 1-desamino[9-(biotinylphenylalanine)]AVT, respectively. Half-maximal hydroosmotic responses (ED-50 values) were obtained with 2.5 X 10(-9) M for the iodinated analogue, with 0.9 X 10(-10) M for the photoaffinity analogue, and with 1.2 X 10(-8) M for the biotinyl analogue. The hydroosmotic activity of the biotinyl analogue was reversed following addition of avidin, whereas the photoaffinity analogue induced a persistent response following UV irradiation that was not reversed upon repeated and prolonged periods of washout. These analogues of vasotocin are the most potent that have been synthesized to date, and they should serve as useful probes in the isolation and characterization of vasotocin receptors in toad bladders and tissues from other species that use vasotocin as an antidiuretic/pressor principle. The photoaffinity and biotinyl analogues had a rat antidiuretic activity of 66 and 40 units/mg, respectively. These compounds are, therefore, also suitable for the isolation of V-2 vasopressin receptors from mammalian tissues.

Efficient Photoaffinity Labeling of the Rat V1a, Vasopressin Receptor using a Linear Azidopeptidic Antagonist

“…In contrast, the photolabeling method allows covalent labeling of the receptors that have firstly bound the photoprobe. The corresponding molecules can be easily synthesized by introducing a commercial photoactivatable reagent on previously described ligands (Boer and Fahrenholz, 1985;Buku et al, 1989). However, this solution is rarely efficient since the structural modification often results in a strong decrease of the ligand affinity.…”

Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor

“…Consequently, we synthesized PhAcAL(Btn)VP as a specific biotinylated probe for V,, re-ceptors. A biotinylated agonist has been reported for the toad bladder vasotocin receptor (Buku et al, 1989) and Jans et al (1990) synthesized derivatives of [deamino-2,4-diaminobu-tyrate4, Arg'IVP with biotin attached at position 4. These peptides were shown to be agonists and exhibited similar affinity for V,, and V, VPR (Jans et al, 1990).…”

“…The availability of a biotinylated hormone analogue allows the investigator to exploit the very high affinity interaction of biotin with avidin in a variety of immunocytochemical applications (reviewed by Wilchek and Bayer, 1988). Previous investigations have demonstrated that biotinylated analogues of [Arg'IVP and vasotocin, both agonists (Buku et al, 1989;Jans et al, 1990) and an antagonist (Howl et al, 1991a), can bind to neurohypophyseal peptide hormone receptors whilst simultaneously coupling to avidin or its conjugates. An obvious requirement in such studies is that the biotinylated ligand should retain high affinity for its receptor.…”

Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition

“…The success of this approach is critically dependent upon the ability of the biotinyl-substituted hormone ligand to bind simultaneously to both avidin and the receptor. For example, the hydro-osmotic activity of biotinylated analogues of vasotocin was reversed following the addition of avidin [14]. However, the spacer arms of the molecules used in this study were very short.…”