Dissociation of affinity and efficacy in KOR-3 chimeras
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1The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Abstract:
KOR-3 chimeras were constructed in which the first coding exon of KOR-3 was exchanged for the corresponding first coding exon of either MOR-1 (MOR-1/KOR-3) or DOR-1 (DOR-1/KOR-3). All three clones were expressed in CHO cells and characterized with regards to their binding profiles for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies. 125I[Tyr14]OFQ/N labels both KOR-3 (KD 37 pM) and the MOR-1/KOR-3 chimera (KD 39 pM) equally well. Although its affinity for the DOR-1/KOR-3 chimera is quite good (KD 135 pM), it is slightly lower than the other two. Competition studies confirm the high affinity of OFQ/N for all three clones. However, several competitors clearly distinguish the chimeras from KOR-3. OFQ/N(1-11) competes KOR-3 (Ki 55 nM) over 6-fold more potently than either of the chimeras. (Ki values > 350 nM). Conversely, the modest affinity of naloxone benzoylhydrazone for KOR-3 (310 nM) is greatly increased in both the MOR-1/KOR-3 (Ki 69 nM) and DOR-1/KOR-3 (Ki 74 nM) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits forskolin-stimulated cAMP accumulation in both the KOR-3 and the MOR-1/KOR-3 chimera by almost 40%, with IC50 values in the low nanomolar range. Little activity is seen against the DOR-1/KOR-3 chimera. Naloxone benzoylhydrazone inhibits cAMP accumulation in the KOR-3 and the DOR-1/KOR-3 chimera. Although naloxone benzoylhydrazone has higher affinity for the MOR-1/KOR-3 chimera in binding studies than KOR-3 itself, it is inactive in cyclase studies using the MOR-1/KOR-3 chimera, implying that the replacement of the first coding exon increases affinity while decreasing intrinsic activity.
Insights
This study engineered kappa opioid receptor 3 (KOR-3) chimeras to investigate opioid receptor interactions. The first coding exon exchange revealed distinct binding affinities and functional activities for orphanin FQ/nociceptin and naloxone benzoylhydrazone.
Area of Science:
- Pharmacology
- Molecular Biology
- Receptor Binding Assays
Background:
- Opioid receptors play crucial roles in pain modulation and other physiological processes.
- Understanding the structure-activity relationships of opioid receptors, such as kappa opioid receptor 3 (KOR-3), MOR-1, and DOR-1, is vital for developing targeted therapeutics.
- Chimeric receptor constructs allow for the dissection of specific domains responsible for ligand binding and signaling.
Purpose of the Study:
- To investigate the functional roles of the first coding exon of KOR-3 by creating chimeric receptors with MOR-1 and DOR-1.
- To characterize the binding profiles of orphanin FQ/nociceptin (OFQ/N) and various opioids with KOR-3 and its chimeras.
- To assess the functional consequences of these genetic modifications on G-protein coupled receptor signaling, specifically adenylyl cyclase activity.
Main Methods:
- Construction of KOR-3 chimeras by exchanging the first coding exon with that of MOR-1 (MOR-1/KOR-3) or DOR-1 (DOR-1/KOR-3).
- Expression of wild-type KOR-3 and chimeric receptors in Chinese Hamster Ovary (CHO) cells.
- Radioligand binding assays using 125I[Tyr14]OFQ/N and competition studies with various opioid ligands.
- Functional assays measuring forskolin-stimulated cAMP accumulation to assess adenylyl cyclase activity.
Main Results:
- OFQ/N exhibited high affinity for KOR-3, MOR-1/KOR-3, and DOR-1/KOR-3 chimeras, with slightly lower affinity for DOR-1/KOR-3.
- The OFQ/N(1-11) peptide showed significantly lower potency against the chimeras compared to KOR-3, indicating exon 1's role in OFQ/N(1-11) binding.
- Naloxone benzoylhydrazone affinity was significantly increased in both MOR-1/KOR-3 and DOR-1/KOR-3 chimeras compared to KOR-3.
- OFQ/N functionally inhibited cAMP accumulation in KOR-3 and MOR-1/KOR-3, but not DOR-1/KOR-3.
- Naloxone benzoylhydrazone inhibited cAMP in KOR-3 and DOR-1/KOR-3, but was inactive in MOR-1/KOR-3, despite higher binding affinity.
Conclusions:
- The first coding exon of KOR-3 plays a critical role in determining ligand binding selectivity and functional activity, particularly for OFQ/N(1-11) and naloxone benzoylhydrazone.
- Exchanging the first coding exon of KOR-3 with that of MOR-1 or DOR-1 alters receptor pharmacology, impacting both ligand affinity and intrinsic signaling efficacy.
- These findings highlight the importance of specific receptor domains in mediating distinct opioid signaling pathways and provide insights for receptor-based drug design.