Agonist and antagonist modulation of [ 35 S]‐GTPγS binding in transfected CHO cells expressing the neurotensin receptor

1 The functional interaction of the cloned rat neurotensin receptor with intracellular G‐proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [ 35 S]‐GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. 2 The agonist‐induced binding of [ 35 S]‐GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [ 3 H]‐neurotensin to its receptor was inhibited by NaCl. In the presence of 50 m M NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC 50 value of 2.3±0.9 n M ). 3 The stimulation of [ 35 S]‐GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV‐449 (EC 50 value of 1.7±0.4 n M ) and the neurotensin related peptide neuromedin N (EC 50 value of 21±6 n M ). 4 The NT‐induced [ 35 S]‐GTPγS binding was competitively inhibited by SR48692 (pA 2 value of 9.55±0.28), a non‐peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [ 35 S]‐GTPγS. 5 The response to neurotensin was found to be inhibited by the aminosteroid U‐73122, a putative inhibitor of phospholipase C‐dependent processes, indicating that this drug may act at the G‐protein level. 6 Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G‐protein level that is induced by the neuropeptide neurotensin after binding to its receptor.British Journal of Pharmacology (1997) 121 , 1817–1823; doi: 10.1038/sj.bjp.0701334