Morphine-like Activity of Natural Human IgG Autoantibodies Is because of Binding to the First and Third Extracellular Loops of the μ-Opioid Receptor

Gaëtane Macé,Catherine Blanpied,Laurent J Emorine,Philippe Druet,Gilles Dietrich

doi:10.1074/jbc.274.29.20079

Abstract

We have previously demonstrated that randomly selected healthy individuals express anti-human mu-opioid receptor antibodies which behave as agonist in vitro. In this study, we show that the activity of these antibodies was not affected by the deletion of the amino-terminal region of the receptor. Using agarose-bound peptide columns, we affinity-purified IgG specifically directed toward each extracellular loop. Whatever its specificity, each anti-human mu-opioid receptor (hMOR) extracellular loop peptide IgG preparation was unable, when examined individually, to reduce adenylate cyclase activity. Activation of the hMOR was, however, achieved by the simultaneous binding of IgG to the first and third extracellular loops of the receptor. Our results suggest that the simultaneous binding of IgG antibodies to these two loops mimics morphine-induced receptor activation by triggering a coordinated shift of the third and sixth transmembrane helices.

Highlights

EXPERIMENTAL PROCEDURESTransfection—The cDNA encoding the human ␮-opioid receptor (hMOR) and the hMOR lacking the first 61 amino acids in the amino-terminal segment (hMOR⌬1– 61) [8] were inserted into the pcDNA3.1 vector
The presence of low levels of circulating autoantibodies is a normal feature of the immune system
We showed that the morphine-like activity of antibodies present in normal IgG pools [7] is elicited by their simultaneous binding to the first and third extracellular loops of the human ␮-opioid receptor (hMOR)

Summary

EXPERIMENTAL PROCEDURES

Transfection—The cDNA encoding the hMOR and the hMOR lacking the first 61 amino acids in the amino-terminal segment (hMOR⌬1– 61) [8] were inserted into the pcDNA3.1 vector. Binding Assays—Intact cells (3 ϫ 105) were incubated with increasing amounts of [3H]diprenorphine for 60 min at 25 °C. IgG were incubated with confluent hMOR/CHO cells for 1 h at 37 °C. Bound antibodies were eluted using 0.1 M sodium-citrate, pH 3.5, purified by chromatography on protein G-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) and incubated with untransfected CHO cells. Plates were washed before incubation for 60 min at 37 °C with IgG diluted in PBS-gel. Expression and binding properties of wild-type hMOR and NH2terminal domain truncated hMOR⌬1– 61 on transfected CHO cells. Saturation isotherm binding of [3H]diprenorphine was performed for each receptor with 300,000 intact recombinant CHO cells. Ki values for morphine were obtained by displacement of [3H]diprenorphine (1 nM) binding using increasing amounts of morphine ranging from 2 nM to 20 ␮M. Results are expressed as means Ϯ S.E. of three independent experiments performed in duplicates. *, p Ͻ 0.05 (Mann-Withney U test)

83 Ϯ 9 292 Ϯ 11*

RESULTS

DISCUSSION