Sortilin, SorCSIb, and SorLA Vps 10p sorting receptors, are novel γ-secretase substrates

Article (PDF Available)inMolecular Neurodegeneration 1(1):3 · February 2006with38 Reads
DOI: 10.1186/1750-1326-1-3 · Source: PubMed
Abstract
The mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimer's disease brains, interact with ApoE, and modulate Abeta production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75NTR and proNGF. We have investigated and provide evidence for gamma-secretase cleavage of this family of proteins. We provide evidence that these receptors are substrates for presenilin dependent gamma-secretase cleavage. Gamma-secretase cleavage of these sorting receptors is inhibited by gamma-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most gamma-secretase substrates, we find that ectodomain shedding precedes gamma-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an alpha-secretase like cleavage. These data indicate that the alpha- and gamma-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known gamma-secretase substrates, and could possibly regulate the biological functions of these proteins.
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Molecular Neurodegeneration
Open Access
Research article
Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel
γ-secretase substrates
Andrew C Nyborg
1
, ThomasBLadd
1
, Craig W Zwizinski
1
, James J Lah
2
and
Todd E Golde*
1
Address:
1
Department of Neuroscience, Mayo Clinic Jacksonville, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, Florida
32224, USA and
2
Department of Neurology, Center for Neurodegenerative Disease, Emory University, Whitehead Biomedical Research Building,
615 Michael Street, Suite 505, Atlanta, GA 30322, USA
Email: Andrew C Nyborg - nyborg.andrew@mayo.edu; Thomas B Ladd - ladd.thomas@mayo.edu;
Craig W Zwizinski - zwizinski.craig@mayo.edu; James J Lah - jlah@emory.edu; Todd E Golde* - tgolde@mayo.edu
* Corresponding author
Abstract
Background: The mammalian Vps10p sorting receptor family is a group of 5 type I membrane
homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their
luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and
trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be
down regulated in Alzheimer's disease brains, interact with ApoE, and modulate Aβ production.
Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex
of Sortilin, p75
NTR
and proNGF. We have investigated and provide evidence for γ-secretase
cleavage of this family of proteins.
Results: We provide evidence that these receptors are substrates for presenilin dependent γ-
secretase cleavage. γ-Secretase cleavage of these sorting receptors is inhibited by γ-secretase
inhibitors and does not occur in PS1/PS2 knockout cells. Like most γ-secretase substrates, we find
that ectodomain shedding precedes γ-secretase cleavage. The ectodomain cleavage is inhibited by
a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an α-secretase
like cleavage.
Conclusion: These data indicate that the α- and γ-secretase cleavages of the mammalian Vps10p
sorting receptors occur in a fashion analogous to other known γ-secretase substrates, and could
possibly regulate the biological functions of these proteins.
Background
γ-Secretase is a multi-component protease complex com-
prised of Presenilin (PS) 1 or 2 with Aph-1, Pen-2, and
Nicastrin [1,2] that cleaves type I membrane proteins
within their transmembrane domains. γ-Secretase cata-
lyzes a number of important physiological and patho-
physiological cleavages. Following ectodomain cleavage
of the amyloid precursor protein (APP) [3] by β-secretase,
γ-secretase cleavage releases the amyloid beta peptide (Aβ)
that accumulates in the brains of patients with Alzhe-
imer's disease (AD) [4]. γ-Secretase also plays a key role in
mediating signaling via the Notch receptors [5-7]. In most
cases, knockout of presenilin or other components of the
γ-secretase complex produces an embryonic lethal pheno-
Published: 12 June 2006
Molecular Neurodegeneration 2006, 1:3 doi:10.1186/1750-1326-1-3
Received: 26 April 2006
Accepted: 12 June 2006
This article is available from: http://www.molecularneurodegeneration.com/content/1/1/3
© 2006 Nyborg et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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type, that resembles the phenotype produced by knockout
of Notch 1.
To date, more than 25 γ-secretase substrates have been
identified. [8-28]. All identified γ-secretase substrates are
type I transmembrane proteins [29] and contain a puta-
tive stop transfer sequence immediately following the
transmembrane region [30]. In most cases, ectodomain
shedding precedes intramembrane γ-secretase cleavage
[31]. For a growing lists of substrates, γ-secretase cleavage
has been shown to mediate downstream signaling events
[32].
Although many proteases preferentially cleave at consen-
sus sequences within the substrate, no consensus
sequence for cleavage by γ-secretase has been identified. γ-
Secretase cleavage always occurs within a putative hydro-
phobic transmembrane region, but a variety of different
sites are cleaved even in a single substrate. Alignment of γ-
secretase substrate transmembrane domains provides lit-
tle insight into the sequence requirements for proteolysis
to occur (Figure 1a) [33]. Likewise mutations of cleavage
sites have provided little definitive information on the
nature of the cleavage, though several models have been
proposed [34].
Following ectodomain shedding, γ-secretase cleavage lib-
erates both the cytoplasmic fragment and a small secreted
peptide. For several substrates the liberated cytoplasmic
domain has been shown to translocate to the nucleus
where it is involved in nuclear signaling (Notch ErbB4,
Delta-1 Jagged, APLP1/2). This process is more generally
referred to as regulated intramembrane proteolysis (RIP).
RIP of Notch has been intensively studied. Ligand binding
to the Notch extracellular domain results in ectodomain
cleavage, which is then followed by γ-secretase cleavage.
Once liberated the notch intracellular domain (NICD)
translocates to the nucleus where it binds to CSL family of
transcription factors [35,36]. Notch binding to CSL has
been shown to convert CSL from a transcriptional repres-
sor to a transcriptional activator [5]. Analogously nuclear
signaling of the CD44 [37,38] and N-cadherin [39] cyto-
plasmic domains following γ-secretase cleavage is indi-
rectly achieved through CBP (CREB-binding protein)
activation or suppression, respectively. In some cases
nuclear translocation of the cytoplasmic domain is not
required for signaling following γ-secretase cleavage
[8,13,14,40]. Not all γ-secretase substrates appear to
undergo RIP, as ligand binding is not necessary for the ini-
tiation of cleavage. For example, APP does not appear to
require ligand association in order to initiate ectodomain
shedding which occurs prior to γ-secretase cleavage.
In most cases, signaling initiated by γ-secretase cleavage
appears to be an activation event. However, cleavage of
the substrate deleted in colorectal cancer (DCC) attenu-
ates receptor-mediated intracellular signaling pathways
that are critical in regulating glutamatergic synaptic trans-
mission and memory processes [18,41].
Because of their topologic similarity to other γ-secretase
substrates and evidence for ectodomain shedding [42]
(Figure 1a), we hypothesized that the mammalian Vps10p
containing family of proteins might be γ-secretase sub-
strates. Sortilin, SorLA and SorCS1, 2, and 3 comprise the
five identified mammalian Vps10p sorting receptors and
have a number of features in common. First, all are type I
membrane proteins (Figure 1b). Second, all contain a
luminal/extracellular cysteine-rich Vps10p domain
homologous to the binding domain of the yeast sorting
receptor for carboxypeptidase Y. Third, all contain a puta-
tive furin cleavage site. SorCS1-3 and SorLA also contain
additional extracellular domains thought to be involved
in ligand binding (Figure 1b). Sortilin is also known as
neurotensin receptor 3 or gp95, and SorLA is often called
LR11. These receptors are hypothesized to have pleio-
tropic functions in chaperoning and targeting various car-
goes bound to their luminal Vps10p domains between
various intracellular organelles [43,44].
These sorting receptors are expressed at high levels in the
CNS and in neurons [44-46]. Sortilin is part of the
machinery that governs cell survival in developing neuro-
nal tissue and a key determinant in the induction of post-
traumatic neuronal apoptosis [47]. It also mediates rapid
endocytosis of lipoprotein lipase [48], neurotensin [49],
and the proform of nerve growth factor [47]. Sortilin has
been shown to target proteins in the Golgi for transport to
late endosomes. Of its many sorting and signaling func-
tions, Sortilin was shown to play a role in p75
NTR
"death
signaling". The cell death signal is a result of a complex of
p75
NTR
, Sortilin and the precursor form of nerve growth
factor (proNGF) [47] or pro brain-derived neurotrophic
factor [50].
Recent data have suggested a role for SorLA in AD. SorLA
was shown to be a receptor for, and interact with, ApoE
[51-54]. In addition, it is reduced in AD brains versus age
matched controls, interacts with APP [55] and regulates
Aβ production [56,57].
we provide evidence that Sortilin, SorCS1b and SorLA are
sequentially cleaved by an α-secretase like activity fol-
lowed by γ-secretase. α-Secretase cleavage results in secre-
tion of a large extracellular domain of the Vps10p
substrates and γ-secretase liberates a COOH-terminal frag-
ment (CTF) from the membrane. These data extend very
recent studies demonstrating that Sortilin, SorCS1,
SorCS2, SorCS3, and SorLA undergo ectodomain shed-
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ding [42] and that SorLA can undergo γ-secretase cleavage
[28].
Results
Evidence for sequential
α
- and
γ
-secretase cleavage of
Sortilin
HEK 293T cells were transiently transfected with Sortilin
containing a V5 epitope tag at the COOH-terminus
(SorV5) and treated with a variety of inhibitors or PMA.
The epitope tagged SorV5 construct was used because we
have been unable to obtain a reliable anti-sortilin COOH-
terminal antibody. Cell lysates and media from the tran-
siently transfected cells were analyzed by SDS PAGE and
Western blotting. Consistent with previous studies show-
ing that the untagged Sortilin holoprotein is a ~95 kDa
glycoprotein [58], transfection of the SorV5 resulted in
Schematics of γ-secretase substrates and the mammalian Vps10p proteinsFigure 1
Schematics of γ-secretase substrates and the mammalian Vps10p proteins. a) Alignment of the transmembrane
domains and juxtamembrane region of known γ-secretase substrates The number of additional amino acids in the cytoplasmic
domain following the sequence shown is indicated by "+ aa". b) A schematic of the human Vps10p containing proteins is
shown. Sortilin is a 95 kDa glycoprotein. SorCS1, 2, and 3 contain a leucine rich domain and are ~130 kDa. The largest of the
homologs, SorLA is 250 kDa and contains a host of receptor binding domains including 14 different low density lipoprotein
receptor sites, and EGF repeat, and a fribronectin type III repeat.
a
b
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expression of a 95 kDa protein that was detected both by
an anti-Sortilin NH2-terminal antibody (not shown) and
anti-V5 (Figure 2a). In addition, a SorV5 CTF was detected
with the anti-V5 antibody at ~16 kDa (Figure 2a). This
band was not detected by the NH2-terminal Sortilin anti-
body (not shown). γ-Secretase inhibitor treatment mark-
edly increased the ~16 kDa SorV5 CTF in cell lysates of
cells transiently overexpressing SorV5 (Figure 2a), but did
not appreciably alter the amount of soluble Sortilin (sSor)
that was detected in the media with an anti-Sortilin NH2-
terminal antibody but not anti-V5 (Figure 2a). Activation
of protein kinase C by PMA has been shown to enhance
ectodomain shedding of APP by α-secretase [59-62] and
resulted in an increase in sSor detected in the media (Fig-
ure 2a). GM6001, a broad spectrum hydroxamic acid
based inhibitor of matrixmetalloproteases and disin-
tegrin-metalloproteases [63], has been shown to inhibit
α-secretase cleavage of APP and decreased sSor levels
detected in the media. Both GM6001 and PMA treatment
decreased total levels of the 16 kDa SorV5 CTF (Figure 2a).
Anti-β-actin was used to demonstrate loading consistency
between wells.
The increase in 16 kDa sorV5 CTF resulting from γ-secre-
tase inhibition suggested that the SorV5 CTF was a precur-
sor to γ-secretase cleavage. We then expressed SorV5 in
cells lacking PS1 and PS2. Transfection of SorV5 into PS
-/-
MEF cells resulted in an accumulation of the 16 kDa CTF
(Figure 2b). Cotransfection of PS
-/-
MEF with SorV5 and
either PS1 wt or a familial Alzheimer's disease linked
mutant PS1 (M139V) caused the 16 kDa SorV5 CTF band
to almost completely disappear (Figure 2b). Cotransfec-
tion of a dominant negative PS1 aspartate mutant
(D385E) did not reduce levels of the 16 kDa SorV5 CTF.
As expected, PS1 wt and M139V PS1 expression resulted
in generation of endoproteolyzed PS1 CTF (Figure 2b);
and expression of PS1 D385E resulted in accumulation of
the PS1 holo protein (Figure 2b).
Sortilin is processed into a CTF that is increased by γ-secretase inhibition or knockoutFigure 2
Sortilin is processed into a CTF that is increased by γ-secretase inhibition or knockout. a) HEK 293T cells tran-
siently transfected with sorV5 produce a ~95 kDa holoprotein labeled by anti-V5 and anti-Sor NT antibody (not shown). A
~16 kDa CTF of SorV5 is detected by an anti-V5 antibody is present in the cell lysate, and anti-SorNT detects a secreted frag-
ment of ~95 kDa (sSor) that does not label with anti-V5. The anti-V5 positive SorV5 CTF increased with γ-secretase inhibitor
treatment (LY411,575 and compound E) and decreased with either PMA or a metalloprotease inhibitor, GM6001. sSor
increased upon PMA treatment and decreased upon treatment with a metalloprotease inhibitor, GM6001. b) Mouse embry-
onic fibroblasts that are PS
-/-
were transiently transfected with SorV5 plus empty vector, PS1, PS1 M139V, or PS1 D385E. In the
absence of PS1 or the presence of a dominant negative PS1 mutant (D385E) the levels 16 kDa SorV5 CTF are markedly
increased. However, cotransfection of PS1 wt or PS1 familial Alzheimer's disease linked mutant (M139V) and SorV5 lead to an
almost complete reduction in the levels of 16 kDa anti-V5 positive SorV5 CTF. Anti-β-actin was included as a loading control
and anti-PS1ct antibody demonstrated that PS1 wt and M139V underwent endoproteolysis whereas D385E did not.
a
b
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γ
-secretase processing of SorV5 CTF in lipid rafts
In figure 2b the 16 kDa CTF, a putative γ-secretase sub-
strate, increased when no PS was present. Under condi-
tions amenable to γ-secretase cleavage, the 16 kDa band
diminished but a smaller product band was not detected.
It has been demonstrated that γ-secretase activity is
enriched by sucrose gradient fractionation into choles-
terol rich microdomains called lipid rafts [64-67]. To fur-
ther confirm that the 16 kDa SorV5 CTF is a γ-secretase
substrate, cells stably overexpressing SorV5 were lysed in
2% CHAPSO to maintain the γ-secretase complex and
sucrose gradient fractionated as described in the Methods.
Fractions were analyzed by SDS PAGE and Western blot-
ting (Figure 3a). As previously shown, PS1 CTF and NH2-
terminal fragment (NTF) were enriched in the buoyant
fractions 4 and 5 whereas holo PS1 was primarily found
in more dense fractions 10, 11 and 12 (figure 3a). GS27,
a known lipid raft marker, was enriched in the buoyant
fractions as shown previously [64]. However, EEA1, an
early endosomal marker, was found primarily in dense
fractions. The 95 kDa holo SorV5 fractionated to both the
buoyant and dense fractions much like holo APP [64]. A
SorV5 CTF (~16 kDa) was enriched in the buoyant fac-
tions (Figure 3a, "4°C"). Incubation of the all fractions at
37°C for two hours generated a smaller SorV5 CTF (~13
kDa) that was only detected in buoyant fractions (Figure
3 "37°C"). These data provide evidence that the lipid raft
portion of the sucrose gradient contained all the compo-
nents necessary to generate a smaller SorV5 CTF upon
incubation at 37°C.
Having observed the in vitro generation of a smaller SorV5
CTF in fractions 4 and 5 as a result of incubating at 37°C
for 2 hours, we repeated the experiment with cells that
had been pretreated with 50 µM IL-aldehyde, a γ-secretase
inhibitor. These cells were lysed and sucrose gradient frac-
tionated. Buoyant fractions 4 and 5 were combined and
equal aliquots were incubated at 37°C for two hours plus
and minus γ-secretase inhibitors. During the same period
a negative control was incubated at 4°C for two hours. As
shown in Figure 3a, when the buoyant fractions were
incubated at 37°C for two hours a smaller SorV5 CTF
band was generated (figure 3b DMSO). In the sample that
remained at 4°C during the incubation period or those
incubated in the presence of γ-secretase inhibitors no
smaller SorV5 CTF was detected (Figure 3b). Furthermore,
generation of the smaller SorV5 CTF was inhibited by
another γ-secretase inhibitor (LY411,575) in a dose
dependant fashion (Figure 3c). Anti-PS1NT antibody was
used to demonstrate loading consistency.
SorCS1b, evidence for
α
and
γ
-secretase processing of a
second mammalian Vps10p
To further establish that the mammalian Vps10p contain-
ing family members are processed by α and γ-secretase
similar experiments were performed with SorCS1b tagged
with V5 at the COOH-terminus (SorCS1bV5). Consistent
with previous studies showing that the untagged SorCS1b
or His tagged holoprotein was a ~130 kDa [68], transfec-
tion of the SorCS1bV5 resulted in expression of a 130 kDa
protein that was detected by anti-V5 (figure 4a). Transfec-
tion of SorCS1bV5 into PS
-/-
MEF cells resulted in an
increase in the 13 kDa CTF (Figure 4a). Cotransfection of
PS
-/-
MEF with SorCS1bV5 and either PS1 wt or a familial
Alzheimer's disease linked mutant PS1 (M139V) caused a
decrease in the 13 kDa SorV5 CTF band (Figure 4a).
Cotransfection of a dominant negative PS1 aspartate
mutant (D385E) did not reduce levels of the 13 kDa
SorCS1bV5 CTF. Sucrose gradient fractionation of a cell
lysate from HEK cells stably overexpressing SorCS1bV5
lead to an enrichment of a ~13 kDa SorCS1bV5 CTF in
buoyant fractions at 4 and 5 (Figure 4b). Incubating the
fractions at 37°C for two hours generated a faintly detect-
able anti-V5 positive band at ~10 kDa. More consistent
than the detection of this band, was a decrease in the 13
kDa SorCS1bV5 CTF band observed after incubation at
37°C for two hours (Figure 4c and 4d). The decrease in
the ~13 kDa SorCS1bV5 band was inhibited by both
Compound E (Figure 4c) and LY411,575 in a concentra-
tion dependent fashion (Figure 4d). Detection of the sol-
uble SorSC1b NH2-terminus in the media was not
possible due to the lack of a reliable NH2-terminal anti-
SorCS1 antibody.
PS dependant SorLA processing
Using a wt SorLA construct and a COOH-terminal anti-
SorLA antibody (anti-SorLAct) we have obtained evidence
that that SorLA is a γ-secretase substrate as well. The SorLA
holoprotein was detected by the SorLA COOH-terminal
antibody at 250 kDa (Figure 5) consistent with previous
reports [69]. Transfection of SorLA into PS
-/-
MEF cells
resulted in accumulation of a 15 kDa SorLA CTF detected
by an anti-SorLAct antibody (Figure 5). However, cotrans-
fections with SorLA and either PS1 wt or an familial
Alzheimer's disease linked mutant PS1 (M139V) caused a
decrease in the 15 kDa SorLA CTF (Figure 5). Cotransfec-
tion of sorLA with a dominant negative PS1 aspartate
mutant (D385E) did not alter levels of the 15 kDa SorLA
CTF.
Discussion
To date, more than 25 γ-secretase substrates have been
identified (Figure 1a). γ-Secretase cleavage of certain sub-
strates mediates normal physiologic signaling. Given the
growing list of substrates, it has been postulated that γ-
secretase may function much like the proteosome of the
membrane assisting in the degradation of type I trans-
membrane proteins by liberating them from the mem-
brane [70]. This notion may be supported by the
promiscuity with which γ-secretase cleaves multiple sub-
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strates at a variety of different sites (Figure 1a). It is possi-
ble that overexpression of type I transmembrane proteins
may result in non-physiologic γ-secretase cleavage. How-
ever, to date, all γ-secretase substrates initially identified
using overexpression studies in eukaryotic cells have
proven to be authentic endogenous substrates. Indeed,
numerous γ-secretase substrates like APP, Notch, and
CD44 have withstood the scrutiny of genetic, biochemi-
Sucrose gradient fractionation of SorV5 overexpressing cells demonstrates enrichment of the 16 kDa CTF in buoyant "lipid raft" fractions and generation of a smaller SorV5 CTFFigure 3
Sucrose gradient fractionation of SorV5 overexpressing cells demonstrates enrichment of the 16 kDa CTF in
buoyant "lipid raft" fractions and generation of a smaller SorV5 CTF. a) HEK cells stably overexpressing sorV5 were
sucrose gradient fractionated and 1 ml fractions were collected from the top. Each fraction was analyzed by SDS PAGE on 12%
XT Bis-Tris SDS PAGE (MES Buffer) and Western blotting. "4°C" were samples that were maintained at 4°C until SDS PAGE
loading buffer was added. "37°C" indicates samples that were incubated at 37°C for 2 hours and then prepared as western
samples. A smaller SorV5 CTF was detected in fractions 4 and 5 of the 37°C incubated sample. Anti-V5 antibody detected the
SorV5 holo protein at 95 kDa, a CTF at 16 kDa and a smaller CTF in the "37°C" lanes 4 and 5. Anti-GS27 was used as a lipid
raft marker (fractions 4–5), Anti-EEA1 is an early endosomal marker that was not found in lipid raft. PS1 CTF and NTF were
enriched in raft fractions 4 and 5. b) HEK cells stably overexpressing sorV5 were pretreated with a γ-secretase inhibitor (IL-
aldehyde) for 30 hours and sucrose gradient fractions 4, 5 were combined and analyzed for generation of the smaller SorV5
CTF. Samples were equally divided and incubated at 37°C (4°C for control) in the absence or presence of γ-secretase inhibi-
tors, LY411575, and Compound E. A smaller SorV5 CTF band was detected as a result of incubation at 37°C but was blocked
by γ-secretase inhibitors. c) Samples were prepared as in b). Generation of the smaller SorV5 CTF was inhibited in a dose
dependant fashion by a γ-secretase inhibitor, LY411,575. Anti-PS1NT demonstrates the loading consistency.
c
a
b
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PS dependent γ-secretase cleavage of SorCS1bV5 in lipid raftsFigure 4
PS dependent γ-secretase cleavage of SorCS1bV5 in lipid rafts. a) Mouse embryonic fibroblasts that are PS
-/-
were
transiently transfected with SorCS1bV5 in the same fashion as figure 3a. Cells transiently transfected with sorCS1bV5 produce
a ~130 kDa holoprotein labeled by anti-V5. In the absence of PS1 or the presence of a dominant negative PS1 mutant (D385E)
the levels 13 kDa SorCS1bV5 CTF are markedly increased. However, cotransfection of PS1 wt or PS1 familial Alzheimer's dis-
ease linked mutant (M139V) and SorCS1bV5 lead to a reduction in the levels of 13 kDa anti-V5 positive SorCS1bV5 CTF. Anti-
β-actin was included as a loading control and anti-PS1ct antibody demonstrated that PS1 wt and M139V underwent endoprote-
olysis whereas D385E did not. b) HEK cells stably overexpressing sorCS1bV5 were sucrose gradient fractionated and 1 ml
fractions were collected from the top. Each fraction was analyzed by SDS PAGE on 12% XT Bis-Tris SDS PAGE (MES Buffer)
and Western blotting. "4°C" were samples that were maintained at 4°C until SDS PAGE loading buffer was added. "37°C" indi-
cates samples that were incubated at 37°C for 2 hours and then prepared as western samples. A faint SorCS1bV5 CTF was
detected in fractions 4 and 5 of the 37°C incubated sample. Anti-V5 antibody detected the SorV5 holo protein at 130 kDa, a
CTF at 13 kDa and a smaller CTF in the "37°C" lanes 4 and 5. Anti-GS27 was used as a lipid raft marker (fractions 4–5), Anti-
EEA1 is an early endosomal marker that was not found in lipid raft. PS1 CTF and NTF were enriched in raft fractions 4 and 5.
c) HEK cells stably overexpressing sorCS1bV5 were pretreated with a γ-secretase inhibitor (IL-aldehyde) for 30 hours and
sucrose gradient fractions 4, 5 were combined and analyzed for generation of the smaller SorCS1bV5 CTF. Samples were
equally divided and incubated at 37°C (4°C for control) in the absence or presence of γ-secretase inhibitors, LY411575, and
Compound E. A smaller SorV5 CTF band was not detected as a result of incubation at 37°C but the disappearance of the 13
kDa SorCS1bV5 CTF was blocked by γ-secretase inhibitors. d) Samples were prepared as in b). Disappearance of the
SorCS1bV5 CTF was inhibited in a dose dependant fashion by a γ-secretase inhibitor, LY411,575. Anti-PS1NT demonstrates
the loading consistency.
a
c
d
b
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cal, animal model data that establish both their authentic-
ity as true γ-secretase substrates and the physiologic
relevance of γ-secretase cleavage.
Sortilin, SorCS1b and SorLA are all members of the mam-
malian Vps10p sorting receptor family. In this manuscript
we provide several lines of evidence that these proteins are
γ-secretase substrates. We detect truncated CTF derived
from COOH-terminally V5 epitope tagged Sortilin and
SorCS1b and untagged SorLA. These CTF appear to be
derived from the holoproteins by an α-secretase like activ-
ity. γ-Secretase inhibitors and expression of the proteins in
presenilin knockout cells results in marked accumulation
of these CTF, a phenomena reversed by expression wt or
FAD-linked mutant PS1. These CTF are enriched in buoy-
ant lipid raft fractions where γ-secretase activity resides.
Incubation of SorV5 or SorCS1bV5 CTF containing raft
fractions leads to generation of a smaller cleavage product
in the case of Sortilin and a decrease of the CTF in the case
of SorCS1bV5. The generation of the smaller SorV5 CTF
and the decrease in SorCS1bV5 CTF are inhibited by a γ-
secretase inhibitor. Collectively these data indicate that γ-
secretase has the capacity to cleave overexpressed Sortilin,
SorCS1b and SorLA.
During the preparation of this manuscript evidence was
reported that SorLA was a γ-secretase substrate [28]. Bohm
et al demonstrated that Myc tagged SorLA underwent PS
dependent γ-secretase processing and that following γ-
secretase cleavage the cytoplasmic portion was found in
the nucleus [28]. Combined with our data, demonstrating
that Sortilin, SorCS1b, and SorLa are γ-secretase sub-
strates, these data provide evidence that the entire family
of mammalian Vps10p containing type I proteins may
contain important biologically active signaling peptides
in their COOH-termini.
Studies showing intramembrane γ-secretase cleavage of
endogenous Vps10p sorting receptor will be needed to
show that these proteins are in fact authentic γ-secretase
substrates. As noted before it is unlikely that the overex-
pression studies performed here will be misleading. Given
the complex trafficking, sorting, and signaling functions
mediated by mammalian Vps10p sorting receptors it will
be interesting to determine whether γ-secretase cleavage
regulates the normal function of these proteins. Indeed, it
may be that some of the trafficking deficits in PS deficient
cells could be attributed to the lack of γ-secretase cleavage
of several Vps10p proteins. Though speculative there is
some evidence that Vps10p receptors may play a role in
AD and in neuronal death. SorLA has been shown to be
down regulated in AD, and plays a role in APP trafficking
and Aβ production [56,71]. Neuronal death signaling was
shown to result from a complex of two γ-substrates, Sorti-
lin and p75
NTR
[13,14], in conjunction with proNGF or
proBDNF [47]. Additional studies are needed to clarify the
physiologic and possible pathologic role of γ-secretase
cleavage of mammalian Vps10p sorting receptors.
Materials and methods
DNA constructs
Full length Sortilin and SorCS1b plasmid constructs were
purchased from Origene. A V5 epitope was cloned into
the pCMV6-XL plasmid at the COOH-terminus immedi-
ately preceding the stop codon (SorV5 and SorCS1V5).
SorLA wt plasmid was described previously [57]. PS1 wt,
M139V, and D385E were described previously [34]. All
constructs were verified by sequencing.
DNA transfection of PS
-/-
MEF cells
Mouse embryonic fibroblasts (MEF) deficient in both PS1
and PS2 (PS
-/-
) were characterized previously [72]. Effi-
cient transfection of these cells was achieved using the
Amaxa nucleofector system. Briefly, using the Amaxa MEF
PS dependent cleavage of SorLA CTFFigure 5
PS dependent cleavage of SorLA CTF. Mouse embry-
onic fibroblasts that are PS
-/-
were transiently transfected
with SorLA in the same fashion as figure 3a. Cells transiently
transfected with SorLA produce a ~250 kDa holoprotein
labeled by anti-V5. In the absence of PS1 or the presence of a
dominant negative PS1 mutant (D385E) the levels 15 kDa
anti-SorLAct positive SorLA CTF markedly increased.
Cotransfection of PS1 wt or PS1 familial Alzheimer's disease
linked mutant (M139V) and SorLA wt lead to a reduction in
the levels of 15 kDa anti-SorLA positive SorLA CTF. Anti-β-
actin was included as a loading control and anti-PS1ct anti-
body demonstrated that PS1 wt and M139V underwent
endoproteolysis whereas D385E did not.
Molecular Neurodegeneration 2006, 1:3 http://www.molecularneurodegeneration.com/content/1/1/3
Page 9 of 11
(page number not for citation purposes)
kit 2, 3 µg of DNA and 3 × 10^6 cells/reaction the trans-
fection was performed with the "O-05" mouse neuron
program. Transfected cells were plated on 10 cm plate
with 8 ml of growth media. Cells were harvested after 24
hours.
SorV5 and SorCS1bV5 stable cell lines and culture
Human embryonic kidney (HEK) 293 cells were trans-
fected in reduced serum Opti-Mem (Gibco) with 2 µg of
DNA and 8 µl of Fugene. Cells were incubated for 6 hours
and then allowed to equilibrate in standard growth media
(DMEM 2% fetal bovine serum, 8% normal calf serum,
1% penicillin streptomycin) for 24 hours. Selection anti-
biotic was then added to the cells and maintained
throughout the experiments. Transient expression experi-
ments were performed the same but with HEK 293T cells.
Cells treated with inhibitors were incubated for 18 hours
using the concentration reported and 1% DMSO. phorbol
12-myristate-13-acetate (PMA) treatment was performed
for 4 hours.
γ
-secretase inhibitors
Inhibitors were all generated by the Mayo Clinic Chemis-
try core using published methods for each. All com-
pounds were verified by NMR and Mass Spectroscopy.
Lysis, antibodies, and Western blotting
Cells were lysed in 1% triton × 100 with 1× complete pro-
tease inhibitor (Roche) unless otherwise stated. Cell
lysates were then spun at 14,000 RPM for 2 minutes to
remove nuclei. BioRad XT loading buffer with reducing
solution was added to each sample. SDS-PAGE was per-
formed using BioRad Criterion gel system. 12% Bis-Tris
XT gels were used unless otherwise stated with BioRad
MES buffer. Gels were transferred to Millipore low-fluor
PVDF for 90 minutes and 160 volts. Membranes were
blocked in caesine (0.25%) and phosphate buffered
saline solution and primary antibodies were used at the
reported concentration in the blocking solution overnight
at 4°C. Anti-V5 (Invitrogen) and anti-β-actin antibody
(Sigma) antibodies were used at 1:1000. The anti-PS1 NTF
(A4 from Dr. Paul Fraser) antibody I think) and anti-PS1
CTF (490) were used at 1:1000. The anti-SorLAct antibody
was used at 1:500 [57]. Fluorescent antibodies containing
either the 680 or 800 fluorophore were incubated with the
membrane for 1 hour at room temperature at 1:20,000.
Fluorescently labeled protein detection was performed
using the Odyssey Scanner.
Gradient fractionation
Sucrose gradients were run as described previously [64].
Briefly, cells were washed with 5 mL of ice cold PBS (pH
7.4) and lysed in 2.5 mL of 2% CHAPSO 0.15 M Na Cit-
rate (pH 7.0) with 1× protease inhibitor cocktail (com-
plete PI, Roche). The cleared lysate was then sequentially
diluted with sucrose containing 0.15 M Na Citrate (pH
7.0) so that the final concentration of CHAPSO was
0.25% and sucrose was 45%. Four ml of this homogenate
was then applied to the bottom of the centrifuge tube, and
sequentially overlaid with 4 ml of 0.15 M Na Citrate (pH
7.0), 35% sucrose, 0.25% CHAPSO followed by 4 ml of
0.15 M Na Citrate (pH 7.0), 5% sucrose, 0.25% CHAPSO.
The gradient was centrifuged for 19 hrs at 39,000 rpm in
an SW-41 Ti rotor (Beckman) at 4°C. Following centrifu-
gation, 1 mL fractions were collected from the top of the
gradient.
Abbreviations
Abbreviations: CTF, COOH-terminal fragment; NTF,
NH2-terminal fragment; SorV5, sortilin that contain a
COOH-terminal V5 his epitope tag; SorCS1bV5, SorCs1b
that contain a COOH-terminal V5 his epitope tag; PS,
presenilin; APP, amyloid precursor protein; AD, Alzhe-
imer's disease; RIP, regulated intramembrane proteolysis;
NICD, Notch intracellular domain; CBP, CREBP binding
protein; DCC, deleted in colorectal cancer protein; PMA,
phorbol 12-myristate-13-acetate; anti-SorLA, antibody
specific to the COOH-terminus of SorLA; MEF, mouse
embryonic fibroblasts; proNGF, proform of nerve growth
factor; sSor, soluble NH2-terminal fragment of Sortilin;
HEK, human embryonic kidney cell line; PS
-/-
, MEF cells
that PS1 and PS2 are knocked out
Authors' contributions
ACN and TEG contributed to the conception, design,
analysis and interpretation of the data and were responsi-
ble to the manuscript preparation. ACN, TBL and CWZ
carried out the experiments in this manuscript. JJL pro-
vided critical reagents for these experiments. ACN, TEG,
and JJL all contributed to the interpretation and analysis
of data. All authors read and approved the final manu-
script.
Acknowledgements
We thank members of the Golde Lab for their critical analyses and thought-
ful contributions. This work was supported by the Mayo Foundation and an
NIH/NINDS grant NS39072 to T.E.G.
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    • "This is in line with the notion that small alterations in intracellular trafficking of APP are thought to have a modest impact on Ab generation and probably result as a long-term effect over a lifetime in dramatic Ab accumulation (Hermey 2011; Brunholz et al. 2012), supporting the hypothesis that SorCS1 plays a role in APP targeting. However, apart from the assumption that the genetic association of SorCS1 and AD is explained by an influence of SorCS1 on APP transport, it should be taken into consideration that SorCS1, and, interestingly, also SorLA, are substrates of the alpha and gamma secretase (Hermey et al. 2006; Nyborg et al. 2006). It is still unclear if sorting receptor interaction with APP competes for processing of APP by secretases, or if sorting receptors directly modulate trafficking and activity of secretases. "
    [Show abstract] [Hide abstract] ABSTRACT: Processing of amyloid precursor protein (APP) into amyloid-β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co-immunoprecipitation and co-localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common postendocytic pathway. We introduce functional Venus-tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP-positive transport vesicles contain SorCS1. Co-expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP-positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jun 2015
    • "and subsequently increase the progress of neurodegenerative diseases like AD (Nyborg et al. 2006; Lane et al. 2012). For example, sorLA and SORCS1 mediate retrograde trafficking of APP from the cell surface to Golgi compartments and reduce the amyloidergic processing of APP and production of Ab and decrease the risk of AD (Nyborg et al. 2006; "
    [Show abstract] [Hide abstract] ABSTRACT: Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co-receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6-month-old swedish-amyloid precursor protein/PS1dE9 transgenic mice. Aβ42 enhanced the protein and mRNA expression levels of sortilin in a dose- and time-dependent manner in SH-SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75NTR (P75ECD-FC), or the antibody against the extracellular domain of p75NTR, blocked the up-regulation of sortilin induced by Amyloid-β protein (Aβ), suggesting that Aβ42 increased the expression level of sortilin and mRNA in SH-SY5Y via the p75NTR receptor. Inhibition of ROCK, but not Jun N-terminal kinase, suppressed constitutive and Aβ42-induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ42 oligomer increases sortilin gene and protein expression through p75NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ42 and sortilin in Alzheimer's disease. Sortilin is the co-receptor of p75NTR which signals the cell death induced by Aβ and proneurotrophins. We found that sortilin is increased in the AD brain and up-regulated by Aβ and pro-brain-derived neurotrophic factor (proBDNF). Aβ-induced upregulation of sortilin is mediated by p75NTR and the down-streaming RhoA-ROCK signaling pathway. The Aβ/Sortilinp/75NTR signaling may play a role in the pathogenesis of AD.
    Full-text · Article · Jul 2013
    • "Sortilin was first identified as a 95kDa intracellular sorting receptor that directs movements of newly synthesized proteins such as the unprocessed form of nerve growth factor (proNGF), neurotensin, lipoprotein lipase and pro-brain-derived neurotrophic factor (proBDNF) (Lin et al., 1997; Mazella et al., 1998; Munck Petersen et al., 1999; Nyborg et al., 2006; Nykjaer et al., 2004; Teng et al., 2005). Sortilin was also found to function as a larger 100 kDa receptor for these proteins once exposed on the cell surface, usually in concert with another receptor (Jansen et al., 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20 h and without IL-12 in serum free conditions at 48 h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.
    File · Data · Jun 2013 · Journal of Neurochemistry
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