Receptor binding and G-protein activation by new Met(5)-enkephalin-Arg(6)-Phe(7) derived peptides

Met(5)-enkephalin-Arg(6)-Phe(7) (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a na turally occurring heptapeptide that binds to opioid and non-opioid recognit ion sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide sy nthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Ph e-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe- L-Nle-Arg-Phe (III) and Tyr-Gry-Gly-Phe-L-Nle-Arg-Phe (IV). In this study r eceptor binding characteristics and G-protein activation of MERF and its de rivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [H-3]MERF and [ H-3]naloxone binding sites with the exception of analog (II) which turned t o be substantially less active. The presence of 100 mM NaCl or 100 mu M 5'- guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [H-3]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding in rat and frog brain membranes at concentrations of 10(-9)-10( -5) M. The EC50 values Of analog (I) were the highest in both tissues. Anal og (I) was as effective as MERF in rat brain membranes, but showed lower ma ximal stimulation in frog brain preparation. Again, analog (II) seemed to b e the least efficacious peptide that stimulated [S-35]GTP gamma S binding o nly by 59 %. Specificity of the peptides was further investigated by the in hibition of agonist-stimulated [S-35]GTP gamma S binding in the presence of selective antagonists for the opioid receptor types. The mu-selective anta gonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and n altrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.