RESUMO
To understand how two structurally analogous ligand-receptor systems, the nociceptin/opioid receptor-like 1 (ORL1) and dynorphin A/kappa-opioid receptor 1 (KOR1) systems, achieve selectivity, receptor chimeras were generated and analyzed. Replacing discrete domains located between the N-terminus and top of the third transmembrane helix of the KOR1 by the homologous domains of the ORL1 receptor yields hybrid receptors, which, in comparison with the parent KOR1, display up to 300-fold increased affinity but low sensitivity toward nociceptin, and unaltered (high) affinity and sensitivity toward dynorphin A. These substitutions contribute elements for binding of nociceptin but do not suppress determinants necessary for binding and potency of dynorphin A. More importantly, further replacement in these chimeras of the second extracellular loop with that of the ORL1 receptor fully restores responsiveness to nociceptin without impairing responsiveness to dynorphin A. A bifunctional hybrid receptor has thus been identified that binds and responds to both nociceptin and dynorphin A as efficiently as the ORL1 receptor does to nociceptin and the KOR1 to dynorphin A. Together, these results suggest that distinct peptide activation mechanisms operate in the two receptor systems. In particular, the second extracellular receptor loop appears to be an absolute requirement for activation of the ORL1 receptor by nociceptin, but not for activation of the KOR1 by dynorphin A.
Assuntos
Dinorfinas/metabolismo , Peptídeos Opioides/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Dinorfinas/farmacologia , Peptídeos Opioides/farmacologia , Estrutura Secundária de Proteína , Receptores Opioides/química , Receptores Opioides/genética , Receptores Opioides kappa/genética , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Receptor de Nociceptina , NociceptinaRESUMO
The ability to repair DNA interstrand cross-links may be an important factor contributing to mitomycin C (MMC) and cisplatin cytotoxicities. We have assessed the repair of interstrand cross-links induced by MMC in two MMC-hypersensitive hamster cell mutants and their resistant parental cell line. Using a gene-specific repair assay, we found no evidence for repair of MMC cross-links in either parental or mutant cells, suggesting that persistence of DNA interstrand cross-links is not responsible for the differential toxicity of MMC towards hypersensitive cells. Repair of cisplatin-induced interstrand cross-links was efficient in resistant as well as in mutant cells. Therefore we concluded that a defect in excision repair of interstrand cross-links was not responsible for the cytotoxic effects of MMC and cisplatin in these hypersensitive mutants.
Assuntos
Cisplatino/farmacologia , Reparo do DNA , Mitomicina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas , MutaçãoRESUMO
The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.
