Binding and GTPγS autoradiographic analysis of preproorphanin precursor peptide products at the ORL1 and oploid receptors

Utilizing agonist-stimulated GTPgammaS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [S-35]-GTPgammaS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [S-35]-GTPgammaS binding in a pattern similar to that described for [I-125]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II1-17) had no effect, suggesting that they do not mediate their reported analgesic effects via a G(i/o)-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II1 -17, high concentrations of its C-terminal extension, OFQ II1-28, stimulated [S-35]-GTPgammaS binding in a mu (p) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II1-28 at the cloned ORL1 and V opioid receptors. OFQ II1-28 had no specific binding at either ORL1 or mu opioid receptors at concentrations up to 50 muM. This lack of affinity was not consistent with a mu-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II1-28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the mu opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II1-28 does not bind or stimulate [(35)]-GTPgammaS binding in cells expressing the mu opioid receptor. (C) 2003 Elsevier Science B.V. All rights reserved.