Covalent crosslinking of angiotensin II to its binding sites in rat adrenal membranes.

Abstract

To identify the molecular components of the angiotensin II (All) receptor in the adrenal cortex, we have covalently linked "~I-labeled All ("MI-All) to its binding sites by using the chemical crosslinker disuccinimidyl suberate. Membrane fractions from rat adrenal tissue were incubated with "MI-All, washed, and treated with disuccinimidyl suberate. NaDodSO4/polyacrylamide gel electrophoresis carried out under reducing conditions followed by autoradiography showed that a major polypeptide(s) with an average Mr of 116,000 was covalently tagged with 125I-AII. Proteins Preparation of Adrenal Membranes. Membranes from rat adrenal glands were prepared according to the method ofGlossmann et al (1) with minor modifications. Rats were decapitated and their adrenals were removed and immediately placed in icecold buffer (154 mM NaCl/5 mM sodium phosphate, pH 7.4).All subsequent steps were carried out at 4°C. The adrenals were stripped of fat and their capsules with adherent cells were mechanically removed. For convenience, the capsules and remaining tissue were separately homogenized in 20 mM NaHCO3 (pH 7.4) by using 10 strokes with pestle A followed by 10 strokes with pestle B in a 7-ml Dounce homogenizer (Kontes Glass). Typically, the capsules and remaining tissue from 40 adrenals were homogenized separately in 3 and 14 ml of buffer, respectively. The homogenates were then pooled and centrifuged in an International Equipment PR-6000 centrifuge at 600 x gavg for 10 min. The ensuing supernatants were centrifuged at 80,000 X gavg for 1 hr in a Beckman 50 Ti rotor and the pellets so obtained were suspended in 6 ml of 0.25 M sucrose/i mM EDTA/4 mM sodium phosphate, pH 7.4, using five up and down strokes with pestle B in a 7-ml Dounce homogenizer. Two-milliliter portions of this suspension were layered over each of three step gradients consisting of 31.5%, 38.5%, and 42.5% sucrose (wt/wt) in 1 x 3.5 in. (1 in. = 2.54 cm) tubes of a Beckman SW 27 rotor. All sucrose solutions contained 1 mM EDTA/4 mM sodium phosphate, pH 7.4. The gradients were centrifuged at 53,000 X gavg for 2.5 hr. Fractions from the interface of the 0.25 M and 31.5% sucrose layers were collected with a Pasteur pipette, diluted 1:4 with 20 mM NaHCO3 (pH 7.4), and centrifuged at 80,000 X gavg for 1 hr.Binding of AII to Adrenal Membranes. Adrenal membranes were suspended in 120 mM NaCl/20 mM sodium phosphate, pH 7.4, containing, unless otherwise specified, 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and bovine serum albumin at 1 mg/ml. The suspensions were incubated without or with [Sar',Leu8]AII (20-400 nM as indicated) or unlabeled AII (0.5-50 nM) at 22°C for 15 min.

Oligomerization of Wild Type and Nonfunctional Mutant Angiotensin II Type I Receptors Inhibits Gαq Protein Signaling but Not ERK Activation

“…Previously, AT 1 receptor dimerization has been suggested by using various approaches. 1) Using Angiotensin affinity labeling, or chemical cross-linking experiments, AT 1 receptor complexes with a molecular weight corresponding to a dimeric complex have been identified in transfected cell lines and in several organs such as liver adrenal pituitary and brain (41)(42)(43)(44)(45). 2) High affinity ligand binding has been reconstituted by co-expression of two receptors (AT 1 -K102A and -K199A) that did not bind ligand when expressed individually (17).…”

Receptor-receptor interactions in heteroreceptor complexes: a new principle in biology. Focus on their role in learning and memory

“…The first *Correspondence: kjell.fuxe@ki.se doi: 10.7243/2052-6946-2-6 observations indicating the existence of homodimerization of GPCR were made in 1982 [9,10]. In 1987 homodimerization was found to take place upon epidermal growth factor (EGF) induced stimulation of the epidermal growth hormone receptor [11].…”

Functional interactions between 7TM receptors in the Renin-Angiotensin System—Dimerization or crosstalk?

“…These analyses showed that is was possible to identify AT1R receptor complexes with molecular weights corresponding to both a monomeric and a dimeric form of the receptor. However, these observations were not linked to any functional consequences, and it has been speculated, that the dimers could result from technical artefacts causing protein aggregation (9,23,(49)(50)(51)(52)(53).…”