Interaction of [3H]spiperone with rat striatal dopamine D-2 receptors: Kinetic evidence for antagonist-induced formation of ternary complex

T. K. Chatterjee, C. E. Scott, D. M. Vazquez, R. K. Bhatnagar

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Abstract

The characteristics of [3H]spiperone interactions with rat striatal dopamine D-2 receptor were investigated. Although the association of [3H]spiperone occurred monoexponentially, the pseudo-first order rate constant of association showed a hyperbolic dependence on ligand concentration. The data were therefore analyzed with the assumption of a two-step binding reaction leading to ligand-induced receptor isomerization. For the first equilibrium, the dissociation constant (K(D)) was 1.2 nM, while for the second equilibrium, the association and the dissociation rate constants were 71.6 x 10-3 sec-1 and 0.9 x 10-3 sec-1, respectively. The dissociation rate constant of the overall binding reaction, as determined by inducing the dissociation of [3H]spiperone from its binding sites by 1 μM (+)-butaclamol, was 0.92 x 10-3 sec-1. However, the kinetically derived K(D) (15 pM) of the binding reaction differed significantly from the K(D) (218 pM) obtained from equilibrium binding experiments. This inconsistency between the two K(D) values appeared to have arisen from using different receptor concentrations in deriving kinetic and equilibrium data. The K(D) of the equilibrium binding reaction indeed showed significant variation with the receptor concentrations in an inverse way, implicating the involvement of a third component in the two-step binding reaction to form a high affinity ternary complex rather than a simple ligand induced receptor isomerization. Pretreatment of the membrane with 0.1 mM guanosine 5'-imidodiphosphate [Gpp(NH)p] reduced the affinity of the equilibrium binding reaction to a value (K(D) = 1.2 nM) which corresponded to the kinetically derived K(D) of the first step of the binding reaction, indicating the involvement of a guanine nucleotide-binding protein or G protein in inducing the formation of the high affinity ternary complex. The affinity of the binding reaction in Gpp(NH)p-pretreated membranes, however, increased with the duration of incubation, indicating that the ligand receptor complex still can couple with the G protein even in the presence of Gpp(NH)p. Pretreatment of the membrane with pertussis toxin irreversibly decreased the affinity of the binding reaction without significantly affecting the total number of binding sites, implying the involvement of the G(i) subclass of G protein in the interaction of [3H]spiperone with D-2 receptors. Inhibition of the [3H]spiperone binding by a dopamine receptor agonist, bromocriptine, also yielded a monophasic dose response curve both in the presence and in the absence of Gpp(NH)p. However, dopamine inhibited the binding with a multiphasic dose response curve in the presence as well as in the absence of Gpp(NH)p. Furthermore, dopamine accelerated [3H]spiperone dissociation by infinite dilution and inhibited [3H]spiperone binding noncompetitively, characteristics sharply in contrast to those of bromocriptine. We propose that the dopamine D-2 receptor population in striatum does not exist predominantly in association with G proteins; [3H]spiperone interacts with a homogeneous population of the free form of the receptor, with low affinity to promote receptor coupling with G protein to form a high affinity ternary complex, and this ternary complex, induced by antagonist occupancy of the receptor sites, is not destabilized by Gpp(NH)p. These results also allow us to suggest that the anamolous observations for dopamine inhibition of [3H]-spiperone binding may be mediated, in part, by an interaction of dopamine with a site linked allosterically with [3H]spiperone-binding sites.

Original languageEnglish (US)
Pages (from-to)402-413
Number of pages12
JournalMolecular pharmacology
Volume33
Issue number4
StatePublished - Jan 1 1988

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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