Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

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.

Human secreted proteins SLURP‐1 and SLURP‐2 control the growth of epithelial cancer cells via interactions with nicotinic acetylcholine receptors

“…Cell growth was assayed using the WST‐1 colorimetric test as described elsewhere (Lyukmanova et al ., ,b). WST‐1 (water soluble tetrazolium salt 1; Santa Cruz, Dallas, USA) and 1‐m‐PMS (1‐methoxy‐5‐methylphenazinium methyl sulfate, Santa Cruz) were added to cell in concentrations of 0.25 mM and 5 μM, respectively, for 1 h, and formation of coloured product was measured in the 440–460 nm spectral range using microplate reader (Uniplan, ZAO Pikon, Moscow, Russia).…”

“…Some of these proteins, such as Lynx1 and Lypd6 in the brain, are membrane‐tethered, whereas others such as the secreted Ly6/uPAR proteins SLURP‐1 and SLURP‐2, are secreted by epithelial tissues and probably involved in the development of a number of skin diseases including Mal de Meleda (Allan et al ., ; Perez and Khachemoune, ) and psoriasis (Tsuji et al ., ). For example, SLURP‐1 inhibits proliferation of human oral keratinocytes (Het‐1A cells) via selective interactions with non‐neuronal α7 type nAChRs (α7‐nAChRs) (Arredondo et al ., ; Lyukmanova et al ., ), while SLURP‐2 could either inhibit or promote cell growth via signalling through different subtypes of nAChRs and muscarinic acetylcholine receptors (M receptors) (Lyukmanova et al ., ). Using electrophysiological recordings in Xenopus laevis oocytes, we showed that SLURP‐1 is an antagonist of α7‐nAChRs, while SLURP‐2 inhibits α3β2 and α4β2 nAChRs but, at concentrations below 100 nM, can activate α7‐nAChRs in presence of ACh (Lyukmanova et al ., ,b).…”

“…For example, SLURP‐1 inhibits proliferation of human oral keratinocytes (Het‐1A cells) via selective interactions with non‐neuronal α7 type nAChRs (α7‐nAChRs) (Arredondo et al ., ; Lyukmanova et al ., ), while SLURP‐2 could either inhibit or promote cell growth via signalling through different subtypes of nAChRs and muscarinic acetylcholine receptors (M receptors) (Lyukmanova et al ., ). Using electrophysiological recordings in Xenopus laevis oocytes, we showed that SLURP‐1 is an antagonist of α7‐nAChRs, while SLURP‐2 inhibits α3β2 and α4β2 nAChRs but, at concentrations below 100 nM, can activate α7‐nAChRs in presence of ACh (Lyukmanova et al ., ,b).…”

Proteins and chemical chaperones involved in neuronal nicotinic receptor expression and function: an update

“…The LY6 prototoxin family consists of small modulatory proteins that can interact with, numerous targets, including nAChRs and muscarinic ACh receptors (see Tsetlin, ; Lyukmanova et al, ). We here only report the effect of these proteins on the modulation of nAChR signalling.…”

“…Affinity purification of cortical extracts has revealed that SLURP‐2 can interact with α3, 4, α5, α7, β2, and β4 nAChR subunits and has a broad pharmacological profile. In particular, it inhibits ACh ‐evoked currents of α4β2 and α3β2 ‐nAChRs in oocytes and can enhance or inhibit (at higher concentrations) ACh‐evoked currents of the α7 subtype (Lyukmanova et al, ).…”