Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of [alpha]7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC.sub.50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-[alpha]7-nAChRs antibodies revealed [alpha]7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the [alpha]7-nAChRs. Exposure of Xenopus oocytes expressing [alpha]7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC.sub.50 ~ 1 [mu]M). It was shown that rSLURP-1 binds to [alpha]7-nAChRs overexpressed in GH.sub.4 C.sub.l cells, but does not compete with .sup.125 I-[alpha]-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with [alpha]7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of [alpha]7-nAChRs (mecamylamine, [alpha]-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of [alpha]-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through [alpha]7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.