Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors

Abstract

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

MOP and NOP receptor interaction: Studies with a dual expression system and bivalent peptide ligands

“…Using our novel fluorescent ligands [14,39], we aimed to determine whether MOP and NOP receptors were expressed in close proximity to each other in our co-expression system. A significant overlap in binding of both fluorescent probes on the HEK MOP/NOP cell surface was detected, producing a Pearson coefficient correlation of 0.91 (High level of colocalization).…”

“…N/OFQ ATTO594 (excitation 594 nm; emission 620 nm), and/or Derm ATTO488 (excitation 488 nm; emission 520 nm) were allowed to incubate for 5 min, before coverslips were washed with the ice-cold Krebs. Following wash-off, cells were imaged at the desired wavelength (Derm ATTO488 -488nm, image capture at 520 nm; N/OFQ ATTO594 -594 nm, image captured at 620nm), with the images collected by Nikon C1Si software [14,39]. As both probes are agonists all experiments were undertaken at 4˚C to prevent receptor activation and internalisation.…”

“…Binding and functional activity assays were performed to determine the suitability of De101 as a test ligand. Furthermore, we visualised and determined co-expression using two novel fluorescent ligands, N/OFQ ATTO594 [14] for NOP and Dermorphin ATTO488 for MOP [39]. These imaging experiments were performed both using HEK MOP/ NOP and mouse CA1 hippocampal neurons to determine the potential for receptor co-localisation Ex Vivo.…”

Nociceptin/Orphanin FQ receptor expression in primary human umbilical vein endothelial cells is not regulated by exposure to breast cancer cell media or angiogenic stimuli

“… 23 As a control for MOP receptors, we used HEK cells transfected with and expressing high levels of the human MOP receptor. 22 The absence of MOP receptor mRNA is consistent with the absence of Dermoprhin ATTO488 binding. We had anticipated an angiogenic stimulus-driven translation of NOP mRNA into protein, but there was no N/OFQ ATTO594 binding.…”

“…Images were acquired using a Nikon (Amstelveen, The Netherlands) C1Si confocal microscope (60× immersion oil) at the desired wavelength, as described previously. 22 , 23 Specific binding for Dermorphin ATTO488 was determined using naloxone (25 μM) or unlabelled dermorphin (25 μM) and for N/OFQ ATTO594 with the NOP antagonist SB-612111 (25 μM). 24 Cells were pre-incubated with the ligands for 5 min before adding Dermoprhin ATTO488 100 nM or N/OFQ ATTO594 100 nM.…”