The Calcium Sensor Protein Visinin-like Protein-1 Modulates the Surface Expression and Agonist Sensitivity of the α4β2 Nicotinic Acetylcholine Receptor

Abstract

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast twohybrid library using the large cytoplasmic domain of the ␣4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The ␣4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant ␣4␤2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native ␣4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant ␣4␤2 AChRs up-regulated their surface expression levels ϳ2-fold and increased their agonist sensitivity to acetylcholine ϳ3-fold. The modulation of the recombinant ␣4␤2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of ␣4␤2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.Neuronal nicotinic acetylcholine receptors (AChRs) 1 are members of a gene superfamily of ligand-gated ion channels. In vertebrates, neuronal AChRs are composed of subunits ␣2-␣10 and ␤2-␤4 (for review, see Ref. 1). AChR subunits have a large cytoplasmic domain between their third and fourth transmembrane domain whose amino acid sequence is highly divergent among the various subunits (2). The full functional unit of AChRs, like those of the N-methyl-D-aspartate receptors (3), is likely to include proteins that associate with this large cytoplasmic domain and modulate AChR functions.To identify proteins associated with ␣4 AChRs, we used bait consisting of the large cytoplasmic domain of the ␣4 subunit to screen a mouse brain cDNA yeast two-hybrid library. In this paper, we describe the isolation of VILIP-1, a member of the visinin-like protein family of calcium sensor proteins, by such a screen. VILIP-1 is a member of a superfamily of neuronal calcium sensor proteins. This superfamily has been classified into the five subfamilies termed group I-V. The recoverins belong to group I, the frequenins and neuronal calcium sensor (NCS-1) to group II, the VILIPs, hippocalcin, and neurocalcins to group III, NCS-2 to group IV, and guanylyl cyclase-activating proteins and GC-inhibiting proteins to group V (for review, see Ref. 4).The VILIP family is comprised of three members, VILIP-1, VILIP-2, and VILIP-3 (5-10). The members of this family contain 4 EF-hand motifs, of which only EF-hand 2, 3, and 4 are thought to be functional because EF-hand 1 lacks two oxygencontaining side chain residues crucial for binding calcium. A glycine residue at the second position on the polypeptide chain is myristoylated. Interestingly, within most, but not all members of this calcium sensor protein family, the myristoyl moiety is sequestered and exposed through a rapid conformational change that unmasks it in response to alterations in cellular levels of c...

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

“…The M3-M4 loop of nAChRs mediates important receptor properties such as export from the endoplasmic reticulum (ER) (25,26), targeting to axonal or dendritic compartments (27), and interactions with scaffold and signaling molecules (8,28). To identify proteins that associate with the ␤2-M3-M4 loop, we generated a fusion protein encoding the M3-M4 loop (amino acid residues 326-454 of mouse ␤2) in association with GST [GST-␤2 (M3-M4)], and we used a pulldown strategy to isolate the interactions of GST-␤2 (M3-M4) from mouse brain [supporting information (SI) Fig.…”

“…The movement of receptors within cells requires the activity of molecules such as dynamin, a GTPase that mediates vesicle budding, and clathrin, a major constituent of the vesicle coat (8,25). Several ligand-gated ion channels, such as GABA A , are found to traffic in a dynamin-dependent fashion (43).…”

“…Multiprotein complexes (termed signaling complexes or signalplexes) have been found to associate with receptors such as glutamate N-methyl-D-aspartate (NMDA) and mGluR5 (19,20), glucocorticoid (21), serotonin 5-hydroxytryptamine type 2A and 2C (22), dopamine D1 and D2 (23), and the ATP-gated channel (P2X7) (24). Earlier studies based on yeast two-hybrid screens indicate that the ␣4-nAChR binds the scaffold molecule 14-3-3 (8) and the calcium sensor visinin-like protein 1 (25). In this work, we explore interactions of the ␤2 subunit by using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Such studies have also helped to reveal the role of nAChR-interacting proteins such as 14-3-3 [282,283] and VILIP-1 [284] in regulating cell-surface expression of α4β2 nAChRs. Co-expression studies of muscle nAChRs with the cytoplasmic receptor-associated protein rapsyn (also referred to as '43K protein') have helped to establish the role of rapsyn in nAChR clustering by means of expression studies in both Xenopus oocytes [285] and in transfected cell lines [286].…”

Visinin-like protein 1 regulates natriuretic peptide receptor B in the heart

“…These effects are possibly mediated by actions on membrane receptors and ion channels such as the natriuretic peptide receptor B (NPR-B) and the nicotinic acetylcholine receptor [9,10]. VILIP-1 modulates clathrin-dependent receptor cycling in the central nervous system supporting its general role in membrane trafficking [9].…”