Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V_1a vasopressin receptor

We have designed and synthesized a linear peptide analogue of arginine vasopressin. This peptide, [1‐phenylacetyl, 2‐O‐methyl‐D‐tyrosine, 6‐arginine, 8‐arginine, 9‐lysinamide]vasopressin (PhAcALVP), has a lysinamide residue substituted for the more usual glycinamide at position 9. Derivatization of PhAcALVP at the Nɛ‐lysyl amino group with N‐hydroxysuccinimide esters of aminomethylcoumarin (Mec) and biotin (Btn) produced the bifunctional ligands PhAcAL(Mec)VP and PhAcAL(Btn)VP, respectively. Pharmacological characterization of these peptides revealed that all were high‐affinity V_1a‐selective antagonists. PhAcAL(Btn)VP can simultaneously bind to both the rat liver V_1a receptor and avidin conjugates. Using this strategy, we were able to study the distribution of V_1a receptors on the surface of the rat mammary tumour cell line, WRK‐1. Routine epifluorescent microscopy and confocal image analysis were used to observe the distribution of avidin—Texas‐Red associated with receptor‐bound PhAcAL(Btn)VP. We conclude that PhAcALVP is a useful precursor for the production of hetero‐bifunctional V_1a‐selective ligands. Both PhAcAL‐(Mec)VP and PheAcAL(Btn)VP can be used selectively to probe the V_1a receptor and will be versatile tools for a variety of histocytochemical applications, including receptor localization and purification.