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

Utilizing agonist-stimulated GTPγS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [ 35S]-GTPγS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [ 35S]-GTPγS binding in a pattern similar to that described for [ 125I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II 1–17) 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 II 1–17, high concentrations of its C-terminal extension, OFQ II 1–28, stimulated [ 35S]-GTPγS binding in a mu (μ) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II 1–28 at the cloned ORL1 and μ opioid receptors. OFQ II 1–28 had no specific binding at either ORL1 or μ opioid receptors at concentrations up to 50 μM. This lack of affinity was not consistent with a μ-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II 1–28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the μ 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 II 1–28 does not bind or stimulate [ 35S]-GTPγS binding in cells expressing the μ opioid receptor.