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 (KD) 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 microM (+)-butaclamol, was 0.92 X 10(-3) sec-1. However, the kinetically derived KD (15 pM) of the binding reaction differed significantly from the KD (218 pM) obtained from equilibrium binding experiments. This inconsistency between the two KD values appeared to have arisen from using different receptor concentrations in deriving kinetic and equilibrium data. The KD 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 (KD = 1.2 nM) which corresponded to the kinetically derived KD 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 Gi 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.(ABSTRACT TRUNCATED AT 400 WORDS)