Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation.

Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.