Ligand binding, internalization, degradation and regulation by guanine nucleotides of bombesin receptor subtypes: A comparative study

Recent cloning studies confirm two subtypes of Bn receptors exist, a neuromedin B-preferring receptor (NMB-R) and a gastrin-releasing peptide-preferring receptor (GRP-R). Both subtypes occur widely in GI tract and the CNS; however, in contrast to the GRP-R subtype little is known about the ligand-receptor interactions for the NMB-R. Therefore, in the present study we explored the ligand-receptor interactions including kinetics, stoichiometry, internalization, degradation and regulation by guanine nucleotide binding proteins with the NMB-R and compared it to the GRP-R. The rat glioblastoma C-6 cell line which possess functional NMB-R and 3T3 cells which possess functional GRP-R were used. ¹²⁵I-[d-Tyrg̊]NMB and ¹²⁵I-[Tyr⁴]Bn were prepared using Iodogen and purified on HPLC. At 37g̊C binding of ¹²⁵I-[d-Tyrg̊]NMB to NMB-R or ¹²⁵I-[Tyr⁴]Bn to GRP-R was maximal by 5-15 min and decreased to 60-70% after 60 min. HPLC analysis of the 60 min supernatant showed that > 80% of each tracer was degraded. Addition of proteinase inhibitors had a varied inhibitory effect on degradation with the relative order of potency in C-6 cells being leupeptin > bacitracin > chymostatin > phosphoramidon ≫ bestatin and amastatin and 3T3 cells being bacitracin = phosphoramidon > leupeptin = bestatin > chymostatin > amastatin in 3T3 cells. By HPLC analysis addition of bacitracin prevented the degradation in both cell types. With both receptor subtypes dissociation of bound radioligands was slow, with 70-80% of either ¹²⁵I-[d-Tyrg̊]NMB or ¹²⁵I-[Tyr⁴]Bn remained cell-associated after 60 min suggesting possible peptide internalization. With an acid wash procedure to remove surface bound radioligands, 60% of the C-6 cell-associated ¹²⁵I-[d-Tyrg̊]NMB and 52% of the 3T3 cell-associated ¹²⁵I-[Tyr⁴]Bn were internalized after 30 min at 37°C. With membranes from cells possessing either receptor subtype, the stable guanine nucleotide GPP(NH)P inhibited in a dose-dependent fashion binding of ligands. Computer analysis demonstrated that GPP(NH)P decreased receptor affinity for ligands to both receptor subtypes. These results demonstrated that NMB receptors, similar to GRP receptors and rapidly internalize bound agonists and rapidly degrade agonists. The ligand-receptor interaction is regulated by a guanine nucleotide binding protein for both Bn receptor subtypes.