Chimeric opioid peptides: tools for identifying opioid receptor types.

Abstract

We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the K opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affminty and type selectivity characteristic of its N-terminal sequence. The common Cterminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating For experiments on the expression cloning of opioid receptors we wished to develop a system in which peptide ligands and antibodies against the peptides could be used for affinity purification or for isolation of receptor-bearing cells. (Fig. 1), our model peptide, is, in effect, a fusion product of two peptides-the 17-residue dyn A at the N terminus and the 13-residue dynorphin B (dyn B) at the C terminus, connected by a "bridge sequence" LysArg. dyn-32 itself is a K opioid agonist (7 MATERIALS AND METHODSPeptides and mAbs. dyn-32 and all the chimeric peptides were synthesized on an Applied Biosystems 430A peptide synthesizer. Their purities and sequences were confirmed by HPLC analysis and by sequence analysis on the Applied Biosystems 477A protein/peptide sequencer. mAb 17.M was purified from mouse ascites fluid, by using a rat anti-mouse IgG antibody affinity column (Boehringer Mannheim). mAb 39 was produced in hybridoma cell culture in RPMI 1640 medium (J. R. Scientific, Woodland, CA) with fetal calf serum reduced gradually from 10% to 0%. The supernatant was collected by centrifuging (200 x g, 20C, 10 min) and concentrated on a Diaflo ultrafilter (YM type with 5000-Da limit, Amicon) under N2 pressure. Finally, the antibody was purified by immunoaffinity chromatography as above.Peptides and mAbs were labeled with 1251 by the chloramine-T method. 1251I-labeled peptides were purified on HPLC. 1251I-labeled IgG was purified on a Sephadex G-50 column and then on the rat anti-mouse IgG antibody affinity column.ELISA Assays. In antibody titration (dilution) assays, Microtiter plate (Dynatech) wells were coated with 100 ,ul of 0.1 M acetate buffer (pH 4.5) containing chimeric peptide at 5 ,ug/ml, incubated at 4°C overnight, and then washed three times with Dulbecco's phosphate-buffered saline (PBS) (GIBCO, containing calcium and magnesium)/0.05% Tween 20. One hundred microliters of PBS/0.1% bovine serum albumin (BSA) was added to the wells and incubated overnight at 40C to block uncoated sites. The plates were washed again with PBS/Tween. Different concentrations of mAb Abbreviations: BSA, bovine serum albumin; DAGO,

Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the κ opioid receptor using a soluble protein scaffold

“…In support of such a function, chimeric studies show that the peptide selectivity of one receptor subtype may be transferred to another subtype by swapping the loops . Initial recognition of the peptide DynA is mainly controlled by the second extracellular (EL2) κ receptor loop , whereas the active site, most likely responsible for the nanomolar affinity of DynA to KOR , is located in the transmembrane part of the protein .…”

Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands.

“…In preparation for expression cloning, we had developed a set of modified (13) and chimeric (14) opioid peptides, the latter based on the structure of Dyn-32. Dyn-32 is the 17-residue DynA linked at its C terminus through Lys-Arg to DynB (15).…”