IDENTIFICATION OF RESIDUES IMPORTANT FOR LIGAND-BINDING TO THE HUMAN 5-HYDROXYTRYPTAMINE-1A SEROTONIN RECEPTOR

The functional significance of the conserved amino acids within transm embrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A r eceptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembr ane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n- [H-3]propylamino)tetralin. In contrast, replacement of Asn396 with glu tamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alan ine. Changing the less conserved Ser391 to alanine had no effect on li gand binding. However, replacement of the conserved Ser393 with alanin e reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting de tected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations prob ably did not affect expression or processing of the protein. Important ly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been d escribed previously [Nature (Lond.) 329:75-79 (1987)], and has a chang e in the sequence in transmembrane region IV from ... RPRAL... to ... RRAAA.., which corresponds to the published sequence [J. Biol. Chem. 2 65:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that o f the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.