Impaired G protein coupling of the neurotensin receptor 1 by mutations in extracellular loop 3

• Published in: European journal of pharmacology 2001-12, Vol.433 (1), p.63-71
• Main Authors: Richard, Françoise, Barroso, Séverine, Nicolas-Ethève, Delphine, Kitabgi, Patrick, Labbé-Jullié, Catherine
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Biological and medical sciences; cAMP; Cell receptors; Cell structures and functions; COS Cells; Cyclic AMP - biosynthesis; Fundamental and applied biological sciences. Psychology; G protein-coupled receptor; GTP-Binding Proteins - chemistry; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Inositol phosphate; Inositol Phosphates - metabolism; Molecular and cellular biology; Mutagenesis; Mutation; Neuropeptide receptors; Neurotensin - metabolism; Neurotensin receptor 1; Receptors, Neurotensin - analysis; Receptors, Neurotensin - chemistry; Receptors, Neurotensin - metabolism; Recombinant Proteins - metabolism; Structure-Activity Relationship; Transduction, impaired
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/S0014-2999(01)01496-0

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor. We have shown previously that the NTS1 receptor-binding site of the peptide agonist involved residues in extracellular loop 3 and at the extracellular junction of transmembrane domains 4 and 6. Here, we investigated by site-directed mutagenesis residues in extracellular loop 3 that might be involved in agonist-induced activation of the rat NTS1 (rNTS1) receptor. Wild type and mutated receptors were expressed in COS (African green monkey kidney fibroblasts) cells. Labeled agonist and antagonist binding as well as inositol phosphate and cAMP productions were studied. Compared to the wild type NTS1 receptor, the W339A, F344A, H348A and Y349A mutant receptors exhibited (i) decreased proportion of high over low affinity agonist binding sites, (ii) increased sensitivity of high affinity agonist binding to GTPγS, and (iii) impaired G protein coupling of high affinity agonist-receptor complexes. The data are consistent with the C-terminal part of extracellular loop 3 being essential for allowing high affinity agonist-NTS1 receptor complexes to couple to G proteins.