Effect of multiple serine/alanine mutations in the transmembrane spanning region V of the D2 dopamine receptor on ligand binding

Three conserved serine residues (Ser(193), Ser(194), and Ser(197)) in transmembrane spanning region (TM) V of the D-2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser --> Ala mutations had no effect on the binding of most antagonists tested, including [H-3]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala(197) mutation showed a decrease in affinity for domperidone, whereas Ala(193) mutants showed an increased affinity for a substituted benzamide, remoxipride, However, dopamine showed large decreases in affinity (>20-fold) for each multiple mutant receptor containing the Ser(193)Ala mutation, and the high-affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D-2 dopamine receptors. The results obtained suggest that Ser(193) interacts with the hydroxyl of S-5-hydroxy-2-dipropylaminotetralin (OH-DPAT) and Ser(197) with the hydroxyl of R-5-OH-DPAT. We predict that Ser(193) interacts with the hydroxyl of R-7-OH-DPAT and the 3-hydroxyl (m-hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D-2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor-G protein coupling.