Binding and internalization of fluorescent opioid peptide conjugates in living cells

The dynamics of agonist-stimulated opioid receptor internalization and traf ficking have been difficult to study in living cells in part because the av ailable probes were inadequate. To overcome this obstacle, six new fluoresc ent opioid peptides were developed. Dermorphin (DERM), deltorphin (DELT), T IPP, and endomorphin were conjugated to BODIPY TR or Alexa Fluor 488, two f luorescent dyes with distinct hydrophobic properties. In membrane binding a ssays the fluorescent conjugates DERM-A488 or -BTR, DELT-A488 or -BTR, and TIPP-A488 displayed good binding affinity and selectivity for mu- and delta -opioid receptor subtypes. Furthermore, the fluorescent conjugates of derm orphin and deltorphin were biologically active as demonstrated by their abi lity to hyperpolarize locus coeruleus neurons (DERM-A488 or -BTR) and inhib it calcium currents in NG108-15 (DELT-A488). Both of these responses were a ntagonized by naloxone. In conjunction with confocal fluorescent microscopy the trafficking of these fluorescent ligands was monitored in real-time. T he internalization of these ligands by mu- and delta -opioid receptors was found to be naloxone-sensitive and temperature-dependent. Interestingly, on ce these ligands were internalized the fluorescent puncta that formed becam e distributed in one of two patterns. In Chinese hamster ovary cells hetero logously expressing either mu- or delta -opioid receptors the intracellular puncta were concentrated in the perinuclear region of the cell, whereas th ey were distributed throughout the cytoplasm in cells derived from either N G108-15 or SH-SY5Y cells. In summary, we have demonstrated that these novel , fluorescent opioid peptide conjugates permit real-time visual tracking of receptor-ligand complexes, including their internalization and trafficking , in living cells.