Interaction of bivalent ligand KDN21 with heterodimeric delta-kappa opioid receptors in human embryonic kidney 293 cells.
KDN21 is a bivalent ligand that contains delta and kappa opioid antagonist pharmacophores linked through a 21-atom spacer. It has been reported that KDN21 bridges delta and kappa receptors that are organized as heterodimers. We have shown previously that when using [(3)H]diprenorphine as radioligand, KDN21 displayed greatly enhanced affinity in this series for coexpressed delta and kappa opioid receptors (CDK). The present study used in vitro expression systems to investigate interactions of members of the KDN series with delta-kappa heterodimers through competition binding using selective ligands and the mitogen-activated protein kinase (MAPK) assay. In this regard, the use of the selective radioligands [(3)H]naltrindole and [(3)H]norbinaltorphimine (nor-BNI) in competition binding studies revealed that KDN21 has much higher affinity than other KDN members for CDK and bound to CDK more selectively relative to mixed delta and kappa opioid receptors or singly expressed delta and kappa opioid receptors. Other experiments revealed that the binding of naltrindole to delta opioid receptors could increase the binding of nor-BNI to kappa opioid receptors and vice versa, suggesting reciprocal allosteric modulation of receptors in the heterodimer. Regarding the selectivity of KDN21 for phenotypic delta and kappa opioid receptors, we investigated the effect of KDN21 on the activation of MAPKs [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] by delta- or kappa-selective agonists. KDN21 inhibited the activation of ERK1/2 by [D-Pen(2),D-Pen(5)]-enkephalin (delta(1)) and bremazocine (kappa(2)) but had no effect on the activation by deltorphin II (delta(2)) and (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide (U69593, kappa(1)). 7-Benzylidenenaltrexone (delta(1)) and bremazocine (kappa(2)) significantly reduced the binding of KDN21 to CDK, whereas naltriben (delta(2)) and U69593 produced no such change. Taken together, these data support the idea that the organization of delta and kappa receptors as heterodimers gives rise to delta(1) and kappa(2) phenotypes.
Xie
Zhihua
Z
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA.
Bhushan
Rashmi G
RG
Daniels
David J
DJ
Portoghese
Philip S
PS
D000595:Amino Acid Sequence; D002460:Cell Line; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D010766:Phosphorylation; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000378:metabolism*
C487409:KDN 21; D008024:Ligands; D009842:Oligopeptides; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D009271:Naltrexone