Investigation of the role of conserved serine residues in the long form of the rat D2 dopamine receptor using site-directed mutagenesis.

Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane-spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand-binding experiments with [3H] spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 microM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist-binding curves in the absence of GTP, when higher and lower affinity agonist-binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.