THE 5TH TRANSMEMBRANE SEGMENT OF THE NEUROMEDIN-B RECEPTOR IS CRITICAL FOR HIGH-AFFINITY NEUROMEDIN-B BINDING

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin r eleasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affini ty NMB binding, four mutant receptors were constructed, in which diffe rent parts of the NMB-R were replaced with the corresponding regions o f the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four N MB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of I-125-[D-Tyr0]NMB di splacement assays using unlabeled NMB for competition indicated that h igh affinity NMB binding was determined by amino acid sequences in tra nsmembrane domain V (TM-V) of the NMB-R. To identify which amino acid( s) in TM-V of NMB-R contributed to high affinity NMB binding, four add itional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids . Three of the mutations, TyrPheLeu220-222 --> PheTyrVal, Ile230 --> V al, and His234 --> Phe, did not affect high affinity NMB binding. The Ile216 --> Ser substitution, however, abolished high affinity NMB bind ing and severely impaired the ability of the mutant receptor to stimul ate NMB-dependent inositol phosphate formation. These results suggest that Ile216 in TM-V of NMB-R may be critical for high affinity NMB bin ding.