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JBC, Vol. 251, Issue 10, 3096-3104, May, 1976

Adipocyte beta-adrenergic receptors. Identification and subcellular localization by (-)-[3H]dihydroalprenolol binding

L. T. Williams, L. Jarett and R. J. Lefkowitz

(--)-[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify binding sites which have the characteristics of beta-adrenergic receptors in membranes from rat adipocytes. The subcellular distribution of the (--)-[3H]Dihydroalprenolol binding sites was examined. The binding sites were predominantly in the plasma membrane fraction, consistent with the proposal that the physiologically significant beta-adrenergic receptors are localized in the adipocyte plasma membrane. Binding of (--)-[3H]dihydroalprenolol to unfractionated adipose membranes was saturable with 0.24 pmol bound/mg of protein at saturation. Half-maximal saturation occurred at 15 nM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (--)-[3H]dihydroalprenolol with its adipocyte receptor. Kinetic analysis of (--)-[3H]dihydroalprenolol binding provided a value of 2.4 X 10(7) M-1 min-1 for the forward bimolecular rate constant, k1. Dissociation of (--)-[3H]dihydroalprenolol was a first order reaction with a rate constant, k2, of 2.94 X 10(-1) min-1. The ratio k2/k1 = 12 nM provides an independent measurement of the KD for the interaction of (--)-[3H]dihydroalprenolol with its receptor which is in good agreement with the values obtained by steady state analysis (12 to 15 nM). Beta-Adrenergic agonists and antagonists competed for the binding sites in unfractionated adipocyte membranes with a typical beta1-adrenergic specificity. The order of potency of agonists was (--)-isoproterenol greater than (--)-norepinephrine congruent to (--)-epinephrine. The beta-adrenergic antagonist, (--)-propranolol, potently competed for the binding sites with a KD of 17 nM. Compounds such as dihydroxyphenylaline, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine which are structurally related to beta-adrenergic agents, but are devoid of beta-adrenergic physiologicl effects in adipocytes, did not compete for the binding sites. Binding was highly stereospecific, the (+) isomers of adrenergic agonists and antagonists requiring 23- to 330-fold higher concentrations to half-maximally inhibit binding than the corresponding (--) stereoisomers. (--)-[3H]Dihydroalprenolol binding was examined highly enriched plasma membrane, mitochondrial, and microsomal (endoplasmic reticulum) fractions of adipocytes. In the presence of 12 nM (--)-[3H]dihydroalprenolol, the specific activity of binding in the plasma membrane fraction was 5-fold higher than that of the mitochondrial fraction and 8-fold higher than that of the microsomal (endoplasmic reticulum) fraction. The specificity and affinity characteristics of the plasma membrane binding sites were found to be virtually identical with those of the unfractionated adipocyte membranes. The observation that (--)-[3H]dihydroalprenolol binding sites are predominantly localized in the plasma membrane fraction suggests the potential usefulness of this ligand as a marker for adipocyte plasma membranes.
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