TY - JOUR
T1 - Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts
T2 - Analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors
AU - Benya, R. V.
AU - Wada, E.
AU - Battey, J. F.
AU - Fathi, Z.
AU - Wang, L. H.
AU - Mantey, S. A.
AU - Coy, D. H.
AU - Jensen, R. T.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1992/12
Y1 - 1992/12
N2 - The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 ± 0.14 nM) and in C6 cells (Ki = 1.90 ± 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22°. Both cell types internalized NMB at similar rates, with >70% of bound ligand being internalized by 60 min at 22°. The nonhydrolyzable guanosine analogue guanosine 5′-(β,γ-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 ± 0.09 nM; C6, 2.09 ± 0.15 nM) and for the GRP analogue (transfected, 1855 ± 140 nM; C6, 2129 ± 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 ± 0.08 nM; C6, 0.11 ± 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
AB - The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 ± 0.14 nM) and in C6 cells (Ki = 1.90 ± 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22°. Both cell types internalized NMB at similar rates, with >70% of bound ligand being internalized by 60 min at 22°. The nonhydrolyzable guanosine analogue guanosine 5′-(β,γ-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 ± 0.09 nM; C6, 2.09 ± 0.15 nM) and for the GRP analogue (transfected, 1855 ± 140 nM; C6, 2129 ± 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 ± 0.08 nM; C6, 0.11 ± 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
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M3 - Article
C2 - 1336112
AN - SCOPUS:0027082327
SN - 0026-895X
VL - 42
SP - 1058
EP - 1068
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 6
ER -