Labeling and Expression of Opioid Receptors Using N-terminal Fusion of Fluorescent Proteins

Le résumé fourni par la source

The most efficient fluorescent based binding assay requires detection of Forster resonance energy transfer (FRET) or Luminescence Resonance Energy Transfer (LRET) between two matched fluorescent ligands in the former or a chelated lanthanide donor and fluorescent acceptor in the latter, one attached to the receptor and one attached to the ligand. Although there is an abundance of fluorescent based functional assays for G-coupled protein receptors (GPCRs) [1,2], there are few fluorescent based receptor binding assays, the only commercial fluorescent based binding assay contains SNAP tags [3,4], although capable of FRET it requires the expression of a heterologous protein which is subsequently labeled with a fluorescent tag. Many of the GPCRs including the opioid receptors have short N-terminal tails, usually 20-50 amino acids. Early attempts to fuse FP to the receptors yielded poor results such that today fusion to the C-terminal tail is the preferred method of labeling receptors. There are notable exceptions such as class II GPCRs that naturally have large extracellular domains such as vasoactive intestinal peptide receptor (VPAC1), these receptors readily accept fusion of a FP to the N-terminal tail [5]. GPCRs with large extracellular domains usually have a cleavable signal peptide that enables cell surface expression. Increasing receptor expression at the cell surface by inserting a cleavable signal sequence to heterologous receptors was first described for adrenergic receptors [6] and is now a widely used method for receptor expression. Early studies described for the mu opioid receptor (MOR) with a FP fused to the N-terminal of the receptor by insertion of the FP between the signal sequence of the insect endogenously expressed Immunoglobulin Heavy Chain Binding Protein (BiP) and MOR. Cell surface expression was achieved but FP-MOR was limited to insect cells and receptors were observed to be trapped within intracellular compartments [7]. In mammalian cells it appeared that insertion of a signal sequence interfered with the natural regulation of expression via glycosylation [8,9] or alternate strategies such as binding to p24A through the acidic residues at the second extracellular loop [10].

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