Abstract

The highly conserved aspartate residue in the second transmembrane domain of G protein-coupled receptors is present in position 113 in the type 1 neurotensin receptor (NTR1) but is replaced by an Ala residue in position 79 in the type 2 neurotensin receptor (NTR2). NTR1 couples to Gαq to stimulate phospholipase C and its binding affinity for neurotensin is decreased by sodium ions and GTP analogs. By contrast, NTR2 does not seem to couple to any G protein in eukaryotic cells, and its binding of neurotensin is insensitive to sodium and GTP analogs. By using site-directed mutagenesis, we substituted Asp113 of the NTR1 by alanine and the homologous residue Ala79 of NTR2 by aspartate. Both mutant receptors display similar affinity for neurotensin as compared with their respective wild type. We demonstrate that the presence of the Asp residue determines by itself the occurrence of the sodium effect on neurotensin affinity for both wild-type and mutated NTR1 and -2. The introduction of an Asp in the second transmembrane domain of NTR2 is not enough to restore a functional coupling to G proteins. In contrast, replacement of Asp113 by Ala residue in NTR1 strongly decreases its ability to activate inositol turnover, indicating that the functionally active conformation of NTR1 is maintained by interaction of sodium ions with aspartate 113.