Solubilized receptors for [3H]dopamine (D3 binding sites) from canine brain

The objective of the present study was to solubilize the D3 site which binds [3H]dopamine, using the same digitonin method that had been successful in solubilizing the D2 dopamine receptor. Canine brain striatal membranes were solubilized by a final concentration of 1% digitonin. The specific binding of [3H]dopamine to the soluble D3 binding sites was measured using Sephadex G-50 gel filtration. The density of D3 sites was identical in the membrane and soluble preparations (82-90 fmoles/mg protein), although the dissociation constant (KD value) went from 1.2 nM in the membranes to the value of 3.4 nM in the soluble material. The concentrations of various drugs which inhibited the binding of [3H]dopamine were similar in the two preparations. The agonists [dopamine, apomorphine and (+/-)-6,7-dihydroxy-2-aminotetralin (ADTN)] all inhibited the binding of [3H]dopamine by 50% at concentrations between 2 and 20 nM in both the intact and soluble preparations. The neuroleptics were all equally weak in inhibiting the binding of [3H]dopamine, with IC50 values in the micromolar concentration range, values typical for the D3 site. Approximately 36% of the D3 sites were recovered from the original tissue. Since the densities and recoveries of the D2 and D3 sites differed upon digitonin solubilization, this provided further indirect evidence that these two sites are distinct and separate entities which might ultimately be separated.