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

• Published in: PloS one 2016-02, Vol.11 (2)
• Main Authors: Lyukmanova, Ekaterina N, Shulepko, Mikhail A, Kudryavtsev, Denis, Bychkov, Maxim L, Kulbatskii, Dmitrii S, Kasheverov, Igor E, Astapova, Maria V, Feofanov, Alexey V, Thomsen, Morten S, Mikkelsen, Jens D, Shenkarev, Zakhar O, Tsetlin, Victor I, Dolgikh, Dmitry A, Kirpichnikov, Mikhail P
• Format: Article
• Language: English
• Subjects: Acetylcholine receptors; Cellular signal transduction; Development and progression; Genetic aspects; Inflammation; Physiological aspects
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0149733

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.