1. Cell type-dependent TMEM35 expression and its effects on surface α7 nicotinic acetylcholine receptors
Brijesh Garg, Hangqing Lin, Ralph Loring, Alexandra Rezvaya, and Bhargav Tilak
Dept. Pharmaceutical Sci., Northeastern Univ., Boston, MA
Abstract Results
Conclusions
References
Surface α7 nicotinic acetylcholine receptor (nAChR) expression depends on the cell type, possibly due to
differences in chaperone proteins that allow assembly and transport to the cell membrane. RIC3 (Resistance
to inhibitors of cholinesterase 3) is a known chaperone for α7 nAChR, but we previously found that it is not
significantly expressed in GH4C1, a rat pituitary cell line that when transfected with α7 nAChR plasmid,
readily produces surface receptors measured by α-bungarotoxin binding (Koperniak et al, J. Neurochem. 124:
300, 2013). Recently, Gu et al. (Neuron 89: 1, 2016) reported that Transmembrane Protein 35 (TMEM35, also
known as NACHO) acts as another chaperone in HEK293 cells when expressed alone with α7 and also
enhances RIC3 effects. We investigated how widely TMEM35 is expressed in cell lines that have little or no
endogenous α7 nAChRs. We compared those cells lines for surface expression of transfected α7. GH4C1 and
GH3 cells readily express surface α7 when transfected, while SH-SY5Y has minor endogenous α7 expression,
and SH-EP1, HEK293, RAW264.7 and H9C2 cells showed no expression when transfected. TMEM35 Western
blots showed a rank order for TMEM35 protein of GH3≥GH4C1>>SH-SY5Y, with no expression in other cells.
These data suggest a correlation between the level of endogenous TMEM35 protein and surface α7
expression in cell lines. However, preliminary binding experiments suggest that TMEM35 tagged with C
terminal GFP does not significantly enhance α7 surface expression in HEK cells transfected with both RIC3
and α7, while Myc-DDK-tagged TMEM35 significantly decreases surface expression. In contrast, TMEM35-
GFP permits significant surface α7 expression in H9C2 cells. If confirmed, these data suggest RIC3 may
interact with the TMEM35 C-terminal.
Background and Methods
Both primary mice macrophages and rat α7 nAChR transfected GH3 cells show 125I-BGT
binding. RAW264.7 and GH3 wild type (WT) do not show 125I-BGT binding (data not
shown for GH3 WT cells). Interestingly, primary macrophages do not express TMEM35
(see above) and very little RIC3, and yet show some surface toxin binding.
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TMEM35-GFP TMEM35-myc-DDK Control
125I-BGTCPMbound/well
Effects of TMEM35 with C-terminal tags on HEK cells
co-transfected with both α7 & RIC3
Ric3 Ab:
a7 a7/S-rRic3 a7/S-rRic3 a7/hRic3 a7/hRic3
+siRNA +siRNA
Non-Permissive SH-EP1 cells Permissive GH4C1 cells
a7 a7/S-rRic3 a7/S-rRic3 a7/hRic3 a7/hRic3
+siRNA +siRNA
Ric3 Ab:
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fmolebound/well
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fmolebound/well
* **
Modified from:
http://www.pdbj.org/pdb_images/2bg9_y.jpg
Assembled a7 receptor
Membrane
Extracellular
space
Intracellular
space
a
a
Receptor assembly in ER
Chaperones?
a
a
a
Chaperones?
a
a
Receptor assembly in ER
Chaperones?Modified from: http://www.med.upenn.edu/
nscience/images2/PromAChRsubtypeswhitea.jpg
a
a
a
Proper folding and
trafficking to
surface
Chaperones?
← also known as TMEM35
E-pub: March 2, 2016
Nicotinic receptor subunits assemble as
pentamers and then traffic to the cell surface
to function. a7 receptors require the
presence of chaperone proteins in the
endoplasmic reticulum to properly assemble.
One known chaperone is RIC3 (Resistance to
Inhibitors of Cholinesterase 3)
Tom Koperniak
Some cell lines, like GH4C1, are permissive for a7 expression (measured by a-bungarotoxin
binding [BGT]) , while others, like SH-EP1, require RIC3 for surface expression.
Our lab (Koperniak et al., J. Neurochem. 124: 300, 2013) recently showed that RIC3 is not
present in permissive GH4C1 cells, and shRNAs and siRNAs that knock down RIC3
expression in these cells have no effect. These results predicted that other a7 receptor
chaperones exist.
Early this year, Gu et al. reported that
Transmembrane Protein 35 (TMEM35-
also known as Novel nAChR regulatOr
or NACHO) is a second a7 receptor
chaperone, and acts alone or
synergistically with RIC3 in non-
permissive cells such as Human
Embryonic Kidney (HEK293).
Methods:
Antibodies: We investigated how widely TMEM35 is expressed in cell lines that have little
or no endogenous α7 nAChRs using a Sigma rabbit polyclonal antibody (catalog
HPA048583) for Western blots.
Cell lines:
GH3 and GH4C1: Rat pituitary-derived cell lines
SH-SY5Y and SH-EP1: Human glioblastoma –derived cell lines
HEK293: Human embryonic kidney cells
RAW264.7: Mouse macrophage-derived cell line
H9C2: Rat cardiac myocyte-derived cell line
Mouse primary macrophages
Plasmids: rat a7 in pRep4, rat RIC3 in pRep4, human TMEME35 –GFP (Origene cat#
RG209790 ) in pCMV6, and human TMEM35-myc-DDK (Origene cat# RC209790 ) in
pCMV6.
Binding assays: Intact cells in 24 well plates incubated in 10 nM 125I-BGT for 3 h at 4⁰ C,
then washed and counted.
Composite data assembled from multiple gels
Western Blots: GH3 cells seem to have the highest TMEM35 expression, followed closely by GH4C1 (in
some experiments similar amounts to GH3), with barely detectible TMEM35 in SH-SY5Y cells and no
detectible TMEM35 in the other cell types tested. This correlates roughly with our experience for
expressing surface a7 receptors in these cells without adding RIC3.
Sample Binding Data: TMEM35 with a C-terminal GFP tag was transfected into rat H9C2 cells
together with a7 receptor plasmid. This combination causes significant surface a7 expression measured
by 125I-BGT binding. Transfection with a7 alone causes no surface expression.
Correlation of TMEM35 expression and a7 surface expression in cell lines
Gu et al. report that TMEM35 is synergistic with RIC3 in HEK293 cells to increase
surface a7 receptor expression. We don’t currently have plasmids for wild type
TMEM35 (without C-terminal tags). In preliminary binding experiments, neither
TMEM35-GFP nor TMEM35-myc-DDK caused significant increases in 125I-BGT toxin
binding, (and the myc-DDK-tagged TMEM35 actually decreased binding) in HEK cells
stably co-transfected with a7 and RIC3. This may possibly indicate that RIC3
interacts in part with the C-terminal of TMEM35.
Synergistic effects of TMEM35 with RIC3?
(data from Koperniak et al. 2013)
• Colombo, S.F., Mazzo, F., Pistillo, F., and Gotti, C. (2013). Biogenesis, trafficking
and up-regulation of nicotinic ACh receptors. Biochem. Pharmacol. 86, 1063–
1073.
• Gu, S., Matta, J.A., Lord, B., Harrington, A.W., Sutton, S.W., Davini, W.B., and
Bredt, D.S. (2016). Brain a7 nicotinic acetylcholine receptor assembly
requires NACHO. Neuron 89, 1–8.
• Halevi, S., Yassin, L., Eshel, M., Sala, F., Sala, S., Criado, M., and Treinin, M.
(2003). Conservation within the RIC-3 gene family. Effectors of mammalian
nicotinic acetylcholine receptor expression. J. Biol. Chem. 278, 34411–34417.
• Koperniak, T.M., Garg, B.K., Boltax, J., and Loring, R.H. (2013). Cell-specific
effects on surface a7 nicotinic receptor expression revealed by over-
expression and knockdown of rat RIC3 protein. J. Neurochem. 124, 300–309.
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RAW264.7 Primary macrophages GH3 rat α7
125I-BGTbinding(CPM/well)
• The level of TMEM35 protein expression in cell lines seems to match the
ability of the cells to express a7 receptor on the cell surface.
• The one exception is mouse primary macrophages, which show surface
toxin binding in the absence of TMEM35
• This may be evidence for another chaperone for a7 receptors on mouse
macrophages, or it may represent expression of a non-a7 receptor that
binds BGT (e.g. a9 or a10).
• TMEM35-GFP acts as a chaperone for a7 in non-permissive cells such as
H9C2 cells.
• However, C-terminal tagged TMEM35 is not synergistic with RIC3 as
reported for untagged TMEM35 in HEK cells.
• A thorough test of this will require plasmids expressing untagged TMEM35,
which are currently under construction.
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GFP Rat α7 Rat α7 + TMEM35-GFP
125I-BGTbinding(CPM/well)
TMEM35-GFP effect on surface a7 expression in rat
H9C2 cardiac cells
Transfection: