Fig 1.
Five biological replicates of both SH-EP1-hα7-Ric-3 cells and SH-EP1-hα7 cells were independently processed and analyzed. Triton X-100 solubilized α7-nAChR protein complexes were isolated from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 extracts using α-bgtx-affinity beads. Binding of α7-nAChRs to affinity beads was confirmed with 125I-α-bgtx radioligand binding assays. Separately, α7-nAChR protein complexes isolated from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 were eluted from affinity beads using 1 M carbachol. Eluted proteins were reduced and alkylated before being digested with trypsin in-solution. Digested peptides from each of the five samples prepared from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cells were analyzed with a Q Exactive mass spectrometer, spectra identified using the Mascot algorithm and results analyzed using ProteoIQ. Identified α7-nAChR associated proteins from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cells were compared. Associations only identified with Ric-3 co-expression in SH-EP1-hα7-Ric-3 cells were determined to be Ric-3-mediated changes in the α7-nAChR interactome.
Fig 2.
125I-α-bgtx binding to affinity immobilized protein.
Detergent solubilized membrane extracts were incubated with α-bgtx-affinity beads for 4 hours at 4°C. Protein-α-bgtx-affinity bead complexes were incubated with 5 nM 125I-α-bgtx for 1 hour at room temperature. Non-specific binding was determined in controls by the inclusion of 1 μM unlabeled α-bgtx to preparations prior to the addition of 125I-α-bgtx. Following incubation with 125I-α-bgtx, beads were washed three times with solubilization buffer and measured. Comparable 125I-α-bgtx binding activity of protein-α-bgtx-affinity bead complexes isolated from SH-EP1-hα7-Ric-3 (56 ± 15 fmol/mg, in blue) and SH-EP1-hα7 (49 ± 9 fmol/mg, in green) was observed (Student’s t test, p = 0.40) while SH-EP1 preparations (in purple) did not show α-bgtx binding activity. No 125I-α-bgtx binding to protein-α-bgtx-affinity bead complexes was observed in samples treated with 5 μM MLA confirming α7-nAChR specificity (Student t test, p < 0.05). SH-EP1-hα7-Ric-3 and SH-EP1-hα7 125I-bgtx binding activity was analyzed with five independent biological replicates. MLA treated samples and SH-EP1 125I-α-bgtx binding activity were analyzed with three independent biological replicates.
Fig 3.
Ric-3 immunoreactivity in SH-EP1-ha7-Ric-3.
Solubilized membrane extracts of SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cell lines were probed with anti-Ric-3 polyclonal antibodies. Ric-3 antibody immunoreactivity at 41 kDa confirms the presence of Ric-3 in membrane extracts from SH-EP1-hα7-Ric-3 cells (A). There is no corresponding band in SH-EP1-hα7 membrane extracts (B). The anti-GAPDH antibody immunoreactivity was utilized as a loading control.
Table 1.
Identification of α7-nAChR in SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cells.
Table 2.
Ontological grouping of Ric-3-mediated α7-nAChR associated proteins.
Table 3.
Summary analysis of Ric-3-mediated proteins with literature citations implicating functional interactions with nAChRs.
Table 4.
Summary of single-peptide-based protein identifications.
Fig 4.
Proteins that could affect the life-cycle of α7-nAChRs.
A total of twenty-one identified proteins have functions that could affect the life-cycle of the α7-nAChR, e.g., receptor biogenesis, modulation of intracellular and plasma-membrane expressed receptor pools, as well as receptor turnover, autophagy, or apoptosis related. These proteins are grouped based on their reported cellular compartment localization. The activity of these proteins may be localized to the endoplasmic reticulum (A), the Golgi complex (B), or the cytosol (C&D). Cytosolic proteins can either be involved in the mobilization of internal pools of α7-nAChRs through kinase and phosphatase activity (C) or be associated with protein turnover, autophagy, and apoptosis-related processes (D).