Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 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 similar to 40%, EC50 similar to 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 noncompetitive inhibition of the response to acetylcholine (up to similar to 70%, IC50 similar to 1 mu M). It was shown that rSLURP-1 binds to alpha 7-nAChRs overexpressed in GH(4)C(l) cells, but does not compete with I-125-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.