The fifth 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 releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane 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 additional 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-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.
Laboratory of Biological Chemistry, Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892.
Z
Z
Fathi
R V
RV
Benya
H
H
Shapira
R T
RT
Jensen
J F
JF
Battey
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015287:Neurokinin B / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D015287:Neurokinin B; C038393:neuromedin B