Efficient Photoaffinity Labeling of the Rat V 1a , Vasopressin Receptor using a Linear Azidopeptidic Antagonist

We have synthesized and fully characterized by fast‐atom‐bombardment‐mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3‐azidophenylpropionyl‐D‐Tyr(Me)‐Phe‐Gln‐Asn‐Arg‐Pro‐Arg‐Tyr(3I)‐NH 2 . Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V 1a receptor [dissociation constant (K d ) = 54+/‐220 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37 , 1841–1849] and for both the rat vasopressin V 1a receptor expressed in Spodoptera frugiperdu 9 cells (Sf9 cells, K d = 688+/−35 pM) and in COS‐7 cells (K d = 320+/−220 pM). This probe labels specifically the V 1a receptors in an ultraviolet‐dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor‐probe associations obtained in Sf9 and COS‐7 cells respectively. These molecular masses are consistent with non‐glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn 2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V 1a receptor and for mapping its antagonist‐binding site.