Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

PLoS One. 2016 Feb 23;11(2):e0149733. doi: 10.1371/journal.pone.0149733. eCollection 2016.

Abstract

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%, EC50 ~ 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-α7-nAChRs antibodies revealed α7 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 α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-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 α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation / drug effects
  • Allosteric Regulation / genetics
  • Antigens, Ly / genetics
  • Antigens, Ly / pharmacology
  • Cell Line
  • Humans
  • Keratinocytes / cytology
  • Keratinocytes / metabolism*
  • Protein Binding
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology
  • Urokinase-Type Plasminogen Activator / genetics
  • Urokinase-Type Plasminogen Activator / metabolism*
  • Urokinase-Type Plasminogen Activator / pharmacology
  • alpha7 Nicotinic Acetylcholine Receptor / antagonists & inhibitors*
  • alpha7 Nicotinic Acetylcholine Receptor / genetics
  • alpha7 Nicotinic Acetylcholine Receptor / metabolism*

Substances

  • Antigens, Ly
  • Recombinant Proteins
  • SLURP1 protein, human
  • alpha7 Nicotinic Acetylcholine Receptor
  • Urokinase-Type Plasminogen Activator

Grant support

The work was partially supported by the Russian Academy of Sciences (Program of Molecular and Cellular Biology), and Russian Foundation of Basic Researches (project № 14-04-00885 issued to VIT, IEK, and DK, and № 16-04-01697 issued to ENL and MAS). The work on affinity purification from cortical extracts and test of antiproliferative activity of rSLURP-1 was done with the support from the Russian Science Foundation (project 14-14-00255 issued to ENL, MAS, MLB, DSK, ZOS, and DAD).