Specific [3H]raclopride binding to neostriatal dopamine D2 receptors: Role of disulfide and sulfhydryl groups

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [³H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compoundN-ethylmaleimide (NEM), produced dose-dependent decreases of specific [³H]raclopride binding; the IC₅₀ values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC₅₀ values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (n_H) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (n_H 0.45–0.64) and both compounds increased the IC₅₀ from 0.7 M (control) to 8 M and 11 M, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (>60%) decrease in the number of high-affinity (R_H) binding. After NEM, there was a decrease in both the number of (R_H) and the affinity (K_H) of the high-affinity binding sites, and in the affinity (K_L) of the low-affinity sites. These results demonstrate the participation of-S-S- and-SH groups in the agonist conformation of theprimary ligand recognition site of the dopamine D2 receptor. Alternatively,-S-S-and-SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type ofG protein.