Pivotal role of an aspartate residue in sodium sensitivity and coupling toG proteins of neurotensin receptors

The highly conserved aspartate residue in the second transmembrane domain o f G protein-coupled receptors is present in position 113 in the type 1 neur otensin receptor (NTR1) but is replaced by an Ala residue in position 79 in the type 2 neurotensin receptor (NTR2). NTR1 couples to Gag to stimulate p hospholipase C and its binding affinity for neurotensin is decreased by sod ium 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 insensitiv e to sodium and GTP analogs. By using site-directed mutagenesis, we substit uted Asp113 of the NTR1 by alanine and the homologous residue Ala79 of NTR2 by aspartate. Both mutant receptors display similar affinity for neurotens in as compared with their respective wild type. We demonstrate that the pre sence 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 no t enough to restore a functional coupling to G proteins. In contrast, repla cement of Asp113 by Ala residue in NTR1 strongly decreases its ability to a ctivate inositol turnover, indicating that the functionally active conforma tion of NTR1 is maintained by interaction of sodium ions with aspartate 113 .