Solubilization and Characterization of Rat Brain α₂‐Adrenergic Receptor

Abstract

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.

Characterization of High‐Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate‐Solubilized Bovine Striatal Preparations

“…Pretreatment of membrane preparations with excess Gpp(NH)p (100 p M ) before solubilization also resulted in the complete loss of [ 3HJNPA binding, thereby implying that the mechanism underlying the conversion of the high-affinity to the low-affinity state of the receptors by guanine nucleotide is identical in membrane and solubilized preparations. The retention of guanine nucleotide sensitivity to the solubilized receptors has also been reported for other systems such as Al- (Stiles, 1985) and a2- (Matsui et al, 1985) adrenergic receptors.…”

Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes

“…It is thought that divalent cations augment agonist binding through interactions with the N, protein (Rodbell, 1980). Because some receptors retain on solubilization only their regulation by guanine nucleotides but not by cations Matsui et a]., 1985) it was assumed that distinct subunits of N, bind guanine nucleotides and cations and that some receptors retain only the guanine nucleotide binding but not the cation binding subunit. In our CHAPS extract the entire regulation by both guanine nucleotides and cations was retained, suggesting that functional receptor-N, complexes were solubilized from rat brain.…”

Opioid Receptors in Magnesium‐Digitonin‐Solubilized Rat Brain Membranes Are Tightly Coupled to a Pertussis Toxin‐Sensitive Guanine Nucleotide‐Binding Protein

“…In the latter case, upon solubilization in the absence of an agonist the high-affinity agonist binding is lost. However, the A, adenosine receptors (Stiles, 1985), the muscarinic receptors (Beme et al, 1984a), dopamine D2 receptors (Kazmi et al, 1986;Senogles et al, 1987), the neurotensin receptors (Mills et al, 1987), and the a2-adrenergic receptors (Matsui et al, 1985;Cerione et al, 1986) have been solubilized in a GTP-sensitive state in the absence of an agonist, consistent with the "disaggregation" theory of Rodbell (1980Rodbell ( , 1985. The fact that a proportion of the opioid receptors are closely associated with the putative Gi and Go proteins, both of which are equally effective in inhibiting adenylate cyclase in reconstituted systems (Cerione et al, 1985), does not answer the question whether either or both confer the biological activity associated with this system.…”