Abstract
Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.
Highlights
Cholinergic signaling in the central and peripheral nervous systems and in non-neuronal tissues including epithelial and immune cells[8]
Initial screening of the optimal conditions for the nuclear magnetic resonance (NMR) study revealed that rSLURP-2 at sub-millimolar concentrations is stable in solution only within a narrow pH range (4.5–6.0)
To reduce protein oligomerization at pH ~5.0, 5% dioxane was added to the sample, which greatly improved the quality of the NMR spectra and permitted studies of the protein spatial structure in solution using the 0.5 mM rSLURP-2 sample (Fig. 1B)
Summary
Cholinergic signaling in the central and peripheral nervous systems and in non-neuronal tissues including epithelial and immune cells[8]. In studies investigating the immortalized line of human oral keratinocytes (cell line Het-1A), SLURP-1 has been shown to inhibit cell proliferation via interactions with α7 nAChRs13,24, while SLURP-2 promotes cell growth. This effect is likely mediated by non-α7 (e.g., α3β2) receptor subtypes[25]. We show that rSLURP-2 acts on a broad range of nAChRs subtypes, on the α3β2 receptor, and we describe a new target of SLURP-2, mAChRs. The overall effect of rSLURP-2 on keratinocyte proliferation likely represents a combination of the contributions from both types of cholinergic signaling. The obtained results revealed some similarities and unique structure-functional properties of SLURP-2 compared with other Ly-6/uPAR proteins, including SLURP-1 and Lynx[1]
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