There is increased interest in the Bn-receptor family because they are 
frequently over/ectopically expressed by tumors and thus useful as targets for 
imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [D-Tyr(6), 
β-Ala(11), Phe(13), Nle(14)]Bn(6-14) [Univ.Lig] has the unique property of 
having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus 
could be especially useful for this approach. However, the molecular basis of 
this property is unclear and is the subject of this study. To accomplish this, 
site-directed mutagenesis was used after identifying potentially important amino 
acids using sequence homology analysis of all BnRs with high affinity for 
Univ.Lig compared to the Cholecystokinin-receptor (CCK(A)R), which has low 
affinity. Using various criteria 74 amino acids were identified and 101 
mutations made in GRPR by changing each to those of CCK(A)R or to alanine. 22 
GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) 
with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing 
>10-fold decrease in Univ.Lig affinity. Additional mutations were made to 
explore the molecular basis for these changes. Our results show that high 
affinity for Univ.Lig by human Bn-receptors requires positively charged amino 
acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 
suggesting charge-charge interactions may be particularly important for 
determining the general high affinity of this ligand. Furthermore, transmembrane 
amino acids particularly in UTM6 are important contributing both charge-charge 
interactions as well as interaction with a tyrosine residue in close proximity 
suggesting possible receptor-peptide cation-π or H-bonding interactions are also 
important for determining its high affinity.