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 mM 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 nM). 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 nM) and the neurotensin related peptide neuromedin N (EC(50) value of 21±6 nM). 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