Abstract

Exposure of bovine caudate homogenates to 53 degrees rapidly (less than 4 min) abolishes subsequent specific binding of the agonist ligand [3H]dopamine to D-3 sites but not that of the butyrophenone dopaminergic antagonist [3H]spiroperidol to D-2 sites in bovine caudate membranes. It has been suggested that this represents selective heat inactivation of the binding site for [3H]dopamine. However, the binding of the agonist [3H]apomorphine is decreased with a time course and temperature dependence indistinguishable from that of [3H]dopamine, despite the ability of [3H]apomorphine to label D-2 sites as well as D-3 sites in control membranes. Heat treatment thus appears to mimic the effects on dopaminergic binding of guanine nucleotides, which, rather than causing a reduction in the number of binding sites, radically lowers agonist affinity at both D-2 and D-3 sites while leaving antagonist affinity at these sites unchanged. In addition, heat treatment and maximal guanine nucleotide addition (300 microM GTP) cause identical decreases in the affinities of agonists in the displacement of [3H]spiroperidol, and similar increases in pseudo-Hill coefficient of these displacements. The effects of heat treatment and guanine nucleotides are not additive, suggesting that their effects may involve a common mechanism. Thus, it is strongly suggested that brief exposure to 53 degrees inactivates not the D-3 site itself, but rather a guanine nucleotide binding factor that regulates both D-2 and D-3 agonist affinities and, as in the beta-adrenergic system, is essential for formation of high-affinity agonist/receptor complexes.