Vol. 64, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
8-ADRENERGIC RECEPTORS:
EVIDENCE FOR NEGATIVE COOPERATIVITY
Lee E. Limbird*, Pierre De Meyts+ and Robert J. Lefkowitz* *Departments of Medicine and Biochemistry Duke University Medical Center, Durham, N.C. 27710; +Diabetes Branch, National Institute of Arthritis, Metabolism and Digestive Diseases National Institutes of Health, Bethesda, Maryland 20014 Received
April
28,
1975
SVMMARY: The specific binding of --[3H](-)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled ~-adrenergic receptors. Hill Plots of such binding data yield slopes (nH="Hill Coefficients") less than 1.0, suggesting that negatively cooperative interactions among the $-adrenergic receptors may occur. The existence of such negative cooperativity was ~onfirmed by a direct kinetic method. The dissociation of receptor bound [JH](-)alprenolol was studied under two conditions: i) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (-)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (-)alprenolol increases the rate of [3H](-)alprenolol dissociation, indicating that negatively cooperative interactions among the ~-adrenergic receptor binding sites do occur.
Radioactively labeled hormones and drugs have been used extensively to study the interaction of these agents with their receptors in target tissues. The application of steady state analysis to such binding data has permitted characterization of these hormone-receptor interactions.
In a few cases,
classical steady-state analyses, e.g. Scatchard and Hill plots, have been consistent with a simple bimolecular reaction, H + R ~ - H R ,
(i).
In most
cases, however, Scatchard plots for binding to receptors have displayed a departure from linearity with upward concavity; the slope for Hill plots in these situations is less than 1.0 (e.g. insulin binding) (i). Two interpretations of such binding data are possible: either several classes of receptor sites with discrete affinities are present or the binding process is negatively cooperative - or both.
Steady state binding data alone cannot
discriminate between the two models.
However, De Meyts and co-workers, using a
new method based on the study of dissociation kinetics, demonstrated directly that insulin binding to its receptors in lymphocytes (I) and other tissues (2) induces negatively cooperative site-site interactions. Catecholamines bind to the $-adrenergic receptors in plasma
membranes
and elicit most, if not all, of their physiologic effects via stimulation of
Copyright © 19 75 by Academic Press, hw. All rights of reproduction in alo' form reserved.
1160
Vol. 64, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
the membrane-bound adenylate cyclase and generation of cAMP.
[3H](-)alprenolol I,
a competitive B-adrenergic antagonist, has recently been documented to bind to sites with characteristics indistinguishable from those expected for binding to the adenylate cyclase-coupled B-adrenergic receptors (3-5).
Steady state
data for the binding of [3H](-)alprenolol to sites in frog erythrocyte membranes yield Hill plots with a slope n H
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
8-ADRENERGIC RECEPTORS:
EVIDENCE FOR NEGATIVE COOPERATIVITY
Lee E. Limbird*, Pierre De Meyts+ and Robert J. Lefkowitz* *Departments of Medicine and Biochemistry Duke University Medical Center, Durham, N.C. 27710; +Diabetes Branch, National Institute of Arthritis, Metabolism and Digestive Diseases National Institutes of Health, Bethesda, Maryland 20014 Received
April
28,
1975
SVMMARY: The specific binding of --[3H](-)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled ~-adrenergic receptors. Hill Plots of such binding data yield slopes (nH="Hill Coefficients") less than 1.0, suggesting that negatively cooperative interactions among the $-adrenergic receptors may occur. The existence of such negative cooperativity was ~onfirmed by a direct kinetic method. The dissociation of receptor bound [JH](-)alprenolol was studied under two conditions: i) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (-)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (-)alprenolol increases the rate of [3H](-)alprenolol dissociation, indicating that negatively cooperative interactions among the ~-adrenergic receptor binding sites do occur.
Radioactively labeled hormones and drugs have been used extensively to study the interaction of these agents with their receptors in target tissues. The application of steady state analysis to such binding data has permitted characterization of these hormone-receptor interactions.
In a few cases,
classical steady-state analyses, e.g. Scatchard and Hill plots, have been consistent with a simple bimolecular reaction, H + R ~ - H R ,
(i).
In most
cases, however, Scatchard plots for binding to receptors have displayed a departure from linearity with upward concavity; the slope for Hill plots in these situations is less than 1.0 (e.g. insulin binding) (i). Two interpretations of such binding data are possible: either several classes of receptor sites with discrete affinities are present or the binding process is negatively cooperative - or both.
Steady state binding data alone cannot
discriminate between the two models.
However, De Meyts and co-workers, using a
new method based on the study of dissociation kinetics, demonstrated directly that insulin binding to its receptors in lymphocytes (I) and other tissues (2) induces negatively cooperative site-site interactions. Catecholamines bind to the $-adrenergic receptors in plasma
membranes
and elicit most, if not all, of their physiologic effects via stimulation of
Copyright © 19 75 by Academic Press, hw. All rights of reproduction in alo' form reserved.
1160
Vol. 64, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
the membrane-bound adenylate cyclase and generation of cAMP.
[3H](-)alprenolol I,
a competitive B-adrenergic antagonist, has recently been documented to bind to sites with characteristics indistinguishable from those expected for binding to the adenylate cyclase-coupled B-adrenergic receptors (3-5).
Steady state
data for the binding of [3H](-)alprenolol to sites in frog erythrocyte membranes yield Hill plots with a slope n H
