Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting

Abstract

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

A New Linear V1AVasopressin Antagonist and Its Use in Characterizing Receptor/G Protein Interactions

“…Incubation of liver membranes with GTP␥S reduces the affinity of the receptor for ligand, presumably by dissociating a receptor/G protein complex (5). A nearly homogeneous preparation of rat liver V 1a receptor has an apparent molecular weight of 58,000, which is in agreement with affinity cross-linking results with crude membrane preparations (6).…”

“…The antibodies reacted with a ϳ60-kDa band from rat liver membranes (Fig. 3), corresponding in size to the purified receptor (5,6). The specific receptor band was absent from comparable amounts of membranes from rat kidney, which contain a preponderance of vasopressin V 2 receptors.…”

“…V 1 receptors are associated with polyphosphoinositide metabolism; V 2 receptor activity is mediated by cAMP. V 1a receptors in liver membranes have been solubilized and characterized (2)(3)(4)(5)(6). Incubation of liver membranes with GTP␥S reduces the affinity of the receptor for ligand, presumably by dissociating a receptor/G protein complex (5).…”

Differential Distribution of Bradykinin B 2 Receptors in the Rat and Human Cardiovascular System

“…34 Freshly removed hearts and vessels were rapidly frozen in liquid nitrogen, and sections of 15 to 20 m were prepared in a cryostat at Ϫ30°C, mounted on slides precoated with poly(lysine) (Sigma), and stored at Ϫ70°C. Before use, the slides were warmed to room temperature and washed with cold 50 mmol/L Tris-HCl, pH 7.4, containing 0.1% bovine serum albumin (Sigma), 1 mmol/L EDTA, 1 mmol/L EGTA, and 20 mol/L captopril …”

Vasopressin and Its Analogues: From Natural Hormones to Multitasking Peptides

“…It also participates in the modulation of behavior in social interactions and in the memory process [ 21 ]. Together with V1a, it activates the phosphatidylinositol hydrolysis pathway (formation of 1,2-diacylglycerol and inositol-1,3,4-triphosphate) and mobilizes intracellular Ca 2+ [ 49 , 54 ]. AVP receptor V2 is found in the kidneys where it is expressed on basolateral surface of renal tubular cells [ 45 , 52 , 55 , 56 ].…”