W1581 The Pharmacophore for Human Bombesin Receptors(BnR) Differs Significantly from That of Rat

Abstract

BKG: The mammalian Bombesin receptors [NMBR and Gastrin-Releasing Peptide receptor (GRPR)] share 50% homology, are both widely distributed in the GI tract/CNS, but their affinities for their natural occurring agonists[NMB ,GRP]markedly differ. Whereas, the basis of GRP's selectivity for GRPR is well-studied, little is known for NMBR. Previous studies suggested NMBR selectivity for NMB depends primarily on differences in the 3rd extracellular region (EC3) of the two receptors, particularly the presence of alanine (A) instead of isoleucine (I) (position 198,GRPR), histidine (H) instead of glutamine (G)(position 202, GRPR) and a serine (S)(position 215,GRPR) for isoleucine in transmembrane region 5 (TM5). However, the effect of these changes in combination or the molecular basis for the difference of affinity caused by these changes is unknown. AIM: To provide insight into effect of those important amino acids in combination and the molecular basis for differences in EC3/adjacent TM5 affecting NMB affinity for NMBR. METHODS: 3 double mutants (loss or gain of NMB affinity) were made in NMBR or GRPR combining amino acid differences in the EC3/TM5 that alone showed effects. 7 EC3 mutants in [H202]GRPR or 5 [I199]NMBR were made, whereH202 or I199were substituted by single amino acids with different chemical properties. Receptors were transiently expressed in Balb-3T3 cells and affinities determined. RESULTS: The [A198I,H202Q]mutation in GRPR increased 5-fold affinity which, was equal to the substitution of the entire EC3 NMBR domain, and greater than each point mutant alone. [A198I,S215]GRPR showed the greatest NMB affinity increase(68-fold) with a complete gain-of-affinity. Replacement of I199 in NMBR by A, which is in the comparable position in GRPR caused a decrease in affinity for NMB(8.6-fold), and the substitution of amino acids with high hydrophobicity(V,L,M) caused minimal changes, whereas amino acids with lower hydrophobicity(E,K) caused a decrease in affinity. Replacing H202 in GRPR with Q, which is the comparable position in NMBR or with other nonpolar amino acid (F,Y,A) caused 1.5-2.5-fold gain in NMB affinity, whereas replacement with other basic amino acids(H202R, H202K) did not increase affinity. CONCLUSIONS: These results show the presence of Ile in NMBR instead of A198(EC3) or S215(TM5) account for most of NMBR's selectivity for NMB, whereas a Q instead H202(EC3) is less important. These results, as well as single mutants studies of different amino acids at these sites, suggest enhanced hydrophobic interactions with ligand at these sites is likely mediating their critical importance for NMB affinity.