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

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.
Ekaterina N Lyukmanova 1 2,  Mikhail A Shulepko 1 2,  Denis Kudryavtsev 3,  Maxim L Bychkov 1 2,  Dmitrii S Kulbatskii 1 2,  Igor E Kasheverov 3,  Maria V Astapova 4,  Alexey V Feofanov 1 4,  Morten S Thomsen 5 6,  Jens D Mikkelsen 6,  Zakhar O Shenkarev 1 4 7,  Victor I Tsetlin 3,  Dmitry A Dolgikh 1 2,  Mikhail P Kirpichnikov 1 2
Affiliations

Affiliations

  • 1 Biological Department, Lomonosov Moscow State University, Moscow, Russian Federation.
  • 2 Department of Bioengineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
  • 3 Department of Molecular Basics of Neurosignalling, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
  • 4 Department of Structural Biology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
  • 5 Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
  • 6 Neurobiology Research Unit, University Hospital, Copenhagen, Copenhagen, Denmark.
  • 7 Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, Russian Federation.
PMID: 26905431 DOI: 10.1371/journal.pone.0149733
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.

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