Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki, PO Box 104, Haartmaninkatu 8, 00251 Helsinki, Finland.
EMBO Reports (Impact Factor: 9.06). 02/2002; 3(1):95-100. DOI: 10.1093/embo-reports/kvf010
Source: PubMed
ABSTRACT
Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

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© 2002 European Molecular Biology Organization EMBO reports vol. 3 | no. 1 | pp 95–100 | 2002 95
Dynamic association of human insulin receptor
with lipid rafts in cells lacking caveolae
Saara Vainio, Sanna Heino, Jan-Eric Månsson
1
, Pam Fredman
1
, Esa Kuismanen
2
,
Outi Vaarala & Elina Ikonen
+
Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki, PO Box 104, Haartmaninkatu 8, 00251 Helsinki,
2
Department of
Biosciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, PO Box 56, 00014 Helsinki, Finland and
1
Institute of Clinical Neuroscience,
teborg University, Sahlgrenska University Hospital/Mölndal, SE 43180 Molndal, Sweden
Received July 4, 2001; revised November 5, 2001; accepted November 16, 2001
Cholesterol-sphingolipid rich plasma membrane domains,
known as rafts, have emerged as important regulators of signal
transduction. The adipocyte insulin receptor (IR) is localized to
and signals via caveolae that are formed by polymerization of
caveolins. Caveolin binds to IR and stimulates signalling. We
report that, in liver-derived cells lacking caveolae, auto-
phosphorylation of the endogenous IR is dependent on raft
lipids, being compromised by acute cyclodextrin-mediated
cholesterol depletion or by antibody clustering of glycosphingo-
lipids. Moreover, we provide evidence that IR becomes
recruited to detergent-resistant domains upon ligand binding
and that clustering of GM2 ganglioside inhibits IR signalling
apparently by excluding the ligand-bound IR from these
domains. Our results indicate that, in cells derived from liver,
an important insulin target tissue, caveolae are not required
for insulin signalling. Rather, the dynamic recruitment of the
ligand-bound IR into rafts may serve to regulate interactions in
the initiation of the IR signalling cascade.
INTRODUCTION
Insulin is an anabolic hormone with a central role in carbo-
hydrate and lipid metabolism and in cell growth. Insulin exerts
its effects via insulin receptor (IR), a transmembrane receptor tyro-
sine kinase forming a stable heterotetramer of two α-subunits
and two β-subunits (White and Kahn, 1994). The extracellular
α-subunits contain the insulin-binding sites, whereas the β-subunits
form the transmembrane and intracellular parts of the receptor,
including the kinase domain. Upon insulin binding, IR under-
goes a conformational change that allows trans-phosphorylation
of the catalytic sites on multiple tyrosine residues (Hubbard
et al., 1994). IR autophosphorylation is needed for receptor
activation. A number of docking proteins, including the insulin
receptor substrates (IRSs), are then tyrosine-phosphorylated by
the active IR, and this creates recognition sites for binding to the
SH2 domains in various signalling proteins (Saltiel, 2001).
The most important tissues for insulin action are liver, adipose
tissue and striated muscle. The concentration of IR is high in
these tissues, with more than 200 000 receptors on adipocytes
and hepatocytes (White and Kahn, 1994). In adipocytes, IRs are
sequestered in caveolar invaginations of the plasma membrane
and are dependent on caveolae for signalling (Gustavsson et al.,
1999). IR has a consensus binding site for interaction with
caveolin (Couet et al., 1997), the cholesterol-binding structural
protein of caveolae, and interaction of IR with caveolin leads to
an increase in IR kinase activity (Yamamoto et al., 1998;
Nystrom et al., 1999). Whereas caveolae are present at high
concentrations on adipocytes and muscle cells, there are rela-
tively few caveolae in hepatocytes (Fielding and Fielding, 2000;
Calvo et al., 2001). Moreover, in hepatocytes, the binding of
insulin to its receptor occurs preferentially on microvilli,
followed by redistribution of the receptor–ligand complex to
non-villous segments of the membrane from where it is internal-
ized via coated pits (Carpentier et al., 1985, 1992).
Increasing evidence suggests that cholesterol-sphingolipid
rich membrane microdomains, known as lipid rafts, play an
important role in signal transduction by functioning as platforms
for signalling receptors (Simons and Toomre, 2000). Because of
their presumably small size (beyond the resolution of light
microscopy), individual rafts are likely to host only a subset of
raft proteins. However, rafts may become clustered and this
further enhances protein interactions, thus triggering or amplifying
signalling (Janes et al., 1999).
+
Corresponding author. Tel: +358 9 4744 8469; Fax: +358 9 4744 8960; E-mail: elina.ikonen@ktl.fi
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96 EMBO reports vol. 3 | no. 1 | 2002
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scientific report
Caveolae represent a subset of rafts (Simons and Toomre,
2000). However, the regulation of specific signalling events by
caveolar lipids is difficult to dissect from the dependence of
caveolar integrity on caveolincholesterol oligomers. In this
work, we have analysed the role of the lipid microenvironment
in the initiation of the insulin signalling cascade in a hepatic cell
line that expresses IRs but lacks caveolae. We demonstrate that
acute changes in cholesterol concentration or local sphingolipid
distribution affect insulin signalling and provide evidence for the
dynamic recruitment of IR into rafts upon activation.
RESULTS
The human hepatoma HuH7 cells express very low levels of
caveolin-1 and caveolin-2 in comparison with differentiated
3T3-L1 adipocytes as judged by western blotting (Figure 1A).
Moreover, no morphologically identifiable caveolae were found
in the HuH7 cell plasma membrane by electron microscopy
(Figure 1B). HuH7 cells express biochemically detectable levels
of endogenous IR and respond metabolically to insulin, e.g. by
increasing lipogenesis (data not shown). Thus, these cells offer a
model system for dissecting the role of non-caveolar raft
domains in insulin signalling.
Cholesterol depletion inhibits IR
and IRS-1 phosphorylation
Cholesterol depletion has been shown to inhibit raft-dependent
signalling (Simons and Toomre, 2000). Therefore, we extracted
cholesterol from HuH7 cells by acute treatment with cyclo-
dextrin and analysed its effect on IR signalling. HuH7 cells were
incubated in the presence of 10 mM methyl-β-cyclodextrin for
15 min at 37°C. This resulted in 50% depletion of cellular
cholesterol as judged by the efflux of [
14
C]cholesterol from
labelled cells (data not shown). During the last 10 min of incub-
ation, the cells were stimulated with insulin. Immunoblotting of
the cell lysates with anti-phosphotyrosine (anti-PY) and anti-IRβ
antibodies revealed that the cholesterol extraction resulted in an
60% decrease in IR phosphorylation in insulin-stimulated cells
(Figure 2). The insulin-dependent tyrosine phosphorylation of
IRS-1 was also impaired by 50%, whereas IRS-2 phosphoryl-
ation was not detectable (Figure 2).
Glycosphingolipid clustering
inhibits IR phosphorylation
Clustering of raft components has been shown to modulate raft-
dependent signalling events. For example, Src-kinase phosphoryl-
ation on the cytosolic side of the membrane is stimulated by
patching of glycosphingolipids or glycosyl phosphatidylinositol
(GPI) anchored proteins on the other side (Horejsi et al., 1998;
Kasahara and Sanai, 1999). We next investigated the effect of
Fig. 1. Caveolin expression in HuH7 cells and 3T3-L1 adipocytes. (A)Cell
lysate (20
µg) was immunoblotted with anti-caveolin-1 and anti-caveolin-2
antibodies. Two exposure times are shown. (B) Electron microscopy of HuH7 cell
and 3T3-L1 adipocyte plasma membrane. Caveolae are abundant in 3T3-L1
adipocytes (arrows) but absent in HuH7 cells.
Fig. 2. Effect of cholesterol depletion on IR and IRS-1 phosphorylation.
(A) HuH7 cells were incubated in the presence (+) or absence (-) of methyl-
β-cyclodextrin (CD) for 5 min. Insulin (ins) was then added to the indicate
d
samples and incubation continued for 10 min. Protein (20 µg) from cell
lysates was analysed by western blotting using anti-PY antibody (i
n
duplicate). The locations of IRS-1, IRS-2 and IR are indicated and their levels
in the same samples shown by western blotting. Incubation with CD alone
also affected protein phosphorylation, as indicated by the appearance of a
n
unknown 160 kDa band in the +CD samples. (B) Quantitation of anti-P
Y
blotting data from three independent experiments. Error bars = SEM (n =46);
*P < 0.05.
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EMBO reports vol. 3 | no. 1 | 2002 97
Insulin signalling without caveolae
scientific report
anti-glycosphingolipid antibody cross-linking on insulin signal-
ling. An anti-ganglioside GM2 IgM antibody was chosen
because HuH7 cells were found to contain high levels of GM2
(0.8 nmol/mg protein). The clustering was performed at 12°C,
according to Harder et al. (1998), by incubating living cells for
1 h in the presence of the primary anti-glycosphingolipid anti-
bodies followed by 1 h in the presence of FITC-conjugated anti-
IgM secondary antibodies. Confocal microscopy revealed that,
after GM2 antibody patching, the staining was distributed in
micron-size clusters (Figure 3A). No clustering of GM2 was
observed when the secondary antibody was incubated after fixa-
tion (Figure 3A). When insulin-dependent tyrosine phosphoryla-
tion of IR was analysed upon anti-GM2 clustering, we found it to
be inhibited by 70% compared to incubation with irrelevant
antibodies (Figure 3B and C). To test whether this effect of
glycolipid clustering is restricted to the hepatoma cells, we
carried out the same experiment using primary mouse hepato-
cytes. Both the patching of GM2 and the parallel reduction in IR
phosphorylation were reproduced in these cells (see Supple-
mentary figure 1 available at EMBO reports Online).
Association of IR with
detergent-resistant membranes
Our observations suggest that an intact raft lipid environment is
important for insulin signalling via IR. To further investigate the
underlying mechanism, we studied the association of IR with
detergent-resistant membranes (DRMs). Triton X-100 insolubility
at 4°C is a commonly used criterion for detecting raft association
of proteins (London and Brown, 2000). To analyse whether IR
associates with DRMs in HuH7 cells, insulin-stimulated and
basal cells were lysed in 0.1% Triton X-100 on ice, and the
lysates were subjected to Optiprep gradient fractionation in the
presence of the detergent. Western blot analysis of the proteins
precipitated from the fractions indicated that the distribution of
IR in the gradient changed in response to insulin (Figure 4A). In
the absence of ligand, the vast majority of the receptors were
found at the bottom of the gradient (i.e. solubilized by the deter-
gent), whereas, in the presence of insulin, a significant fraction
of IR floated to the upper fractions representing DRMs. The non-
raft protein transferrin receptor was completely solubilized by
0.1% Triton X-100 (Figure 4A). Similar results were obtained
when using 1% Triton X-100, although the proportion of IR asso-
ciating with DRMs was slightly smaller (Supplementary figure 2,
and data not shown). These results indicate that, in the
hepatoma cells, IR can partition into detergent-soluble or
detergent-resistant domains, and its association with DRMs
depends on ligand binding. Moreover, we found IR association
Fig. 3. Effect of GM2 clustering on IR phosphorylation. (A) Confocal images
of HuH7 cells stained with anti-GM2 antibodies. Cells were incubated in the
presenceofanti-GM2for1hat12
°C,followedbysecondaryantibody
(patched) or BSA (non-patched) for 1 h at 12
°C. After fixing with PFA, non-
patched cells were stained with the secondary antibody. Lower panels show
the areas marked in the upper panels. (B) Cells were patched with anti-GM2
or irrelevant antibodies (anti LDL-receptor antibody or a mixture of mouse
IgM antibodies) for 1 h at 12
°C, followed by respective secondary antibodies
(1 h at 12
°C), and incubated in the presence or absence of insulin for 3 min a
t
37°C. Cell lysates (20 µg of protein) were analysed by western blotting using
anti-PY antibody. (C) Quantitation of the western blotting data. Error bars =
SEM (n =4);*P <0.05.
Fig. 4. Association of IR with DRMs. (A) Basal and insulin-stimulate
d
(1.5 min) cells were solubilized in 0.1% Triton X-100 on ice and subjected to
TritonOptiprep (OP) gradient fractionation. Equal volume from eac
h
fraction was TCA precipitated and the precipitated proteins analysed by
western blotting using anti-IR
β and anti-transferrin receptor (Tfn-R)
antibodies. (B) Cells were cholesterol depleted and insulin stimulated as i
n
Figure 2. For cholesterol repletion, depleted cells were incubated for 2 h wit
h
CD/cholesterol complex prior to insulin stimulation (10 min). Samples
solubilized in 0.1% Triton X-100 were analysed as in (A), using anti-IR
β
antibodies.
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98 EMBO reports vol. 3 | no. 1 | 2002
S. Vainio et al.
scientific report
with DRMs to be cholesterol dependent, as it was abolished by
the cyclodextrin treatment and recovered after subsequent
cholesterol repletion (Figure 4B).
Effect of glycosphingolipid clustering
on the association of IR with rafts
To test whether glycosphingolipid clustering affects the raft asso-
ciation of IR, we studied the distribution of the receptor in the
TritonOptiprep gradient after antibody patching and insulin
treatment. Immunoblotting with anti-IRβ antibody revealed that,
in cells patched with irrelevant antibodies, the receptor was effi-
ciently recovered from DRMs, whereas, in anti-GM2-patched
cells, the receptor was excluded from them (Figure 5A). When
analysing the tyrosine phosphorylation of IR, the phosphoryl-
ation level in anti-GM2-patched cells was found to be markedly
lower than in control cells (Figure 5A), in accordance with the
previous experiment (Figure 3).
Our data suggest that IR becomes excluded from rafts once
GM2 is clustered and presumably stabilized in them. To test
whether the segregation of IR and GM2 could be observed
morphologically in intact cells, we added FITC-coupled insulin
onto cells in which GM2 had been clustered with antibodies and
visualized the staining by confocal microscopy. We found that
insulin labelling localized preferentially to clusters in the inter-
vening areas of GM2 patches (Figure 5B). In non-patched cells,
insulin gave a similar punctate staining, whereas the GM2
immunoreactivity seemed essentially uniformly distributed,
partially overlapping with that of insulin (Figure 5B). The results
suggest that GM2 clustering is accompanied by segregation of IR
and the glycolipid and that clustering does not inhibit insulin
binding to its receptor.
DISCUSSION
Our results indicate that manipulation of raft lipids (i.e. choles-
terol and sphingolipids) modulate IR signalling in the absence of
caveolae. Furthermore, distinct differences are found between
the caveolae-dependent IR signalling described in adipocytes
and the caveolae-independent signalling, analysed here in
hepatoma cells. First, IR appears to be restricted to caveolae in
adipocytes (Gustavsson et al., 1999), whereas its raft affinity
seems to increase upon ligand binding in hepatoma cells. This
may be related to the finding that IRs were found preferentially
as groups in adipocytes and as single receptors in hepatocytes
(Jarett et al., 1980). Secondly, the removal of cholesterol by
cyclodextrin treatment inhibits IRS-1 phosphorylation without
affecting IR autophosphorylation in adipocytes (Parpal et al.,
2001), whereas IR phosphorylation is also inhibited in hepatoma
cells. It is possible that the lack of caveolae makes IR more
vulnerable to the effects of cholesterol extraction. Interestingly,
we have observed that overexpression of caveolin-1 using
recombinant Semliki Forest virus augments IR phosphorylation
in HuH7 cells (unpublished data), in line with the stimulatory
role of caveolin in insulin signalling observed in other cells
(Yamamoto et al., 1998; Nystrom et al., 1999).
Based on the available IR crystal structure, the constitutively
oligomeric receptor occupies a relatively large area in the plane
of the membrane (~80Å in diameter) (Ottensmeyer et al., 2000).
Insulin binding changes receptor conformation to one that is
Fig. 5. The effect of antibody patching on IR raft association. (A) Following antibody patching as in Figure 3B, cells were treated with insulin for 1.5 min,
solubilized and fractionated as in Figure 4. The distribution of IR and tyrosine-phosphorylated IR was analysed by western blotting. (B) Distribution of GM2 an
d
FITC-insulin in non-patched and anti-GM2-patched cells by confocal microscopy. GM2 staining was performed as in Figure 3, and insulin was added for 5 mi
n
before fixation. Ligand-induced receptor clustering (Jeffrey, 1982) may contribute to the punctate FITC-insulin staining pattern. Insulin preferentially segregates
from clustered GM2 (arrows).
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EMBO reports vol. 3 | no. 1 | 2002 99
Insulin signalling without caveolae
scientific report
permissive for phosphorylation. Although dimerization is not
required for activation, unlike in several other transmembrane
receptor tyrosine kinases, there is considerable evidence for IR
self-aggregation. This has been experimentally observed, e.g. by
IR antibodies (Heffetz and Zick, 1986), and also suggested to be
mediated by the ligand itself (Jeffrey, 1982). According to our
data, the dynamic association of IR with rafts may provide an
additional mode of regulation into this framework. The unoccu-
pied receptor seems to have low affinity for rafts, as assessed by
detergent solubilization, and, upon insulin binding, its raft
affinity increases. It is possible that the nature of the ligand-
bound IR may favour its partitioning into DRMs (independent of
raft association in vivo). However, ligand binding alone is not
sufficient for attaining detergent insolubility, as the ligand-bound
IR can be excluded from DRMs by cholesterol depletion or
glycolipid patching.
Apart from caveolin interaction, the cue conferring IR raft
association could be, for example, fatty acylation as the
membrane spanning β-subunit is palmitoylated (Hedo et al.,
1987; Magee and Siddle, 1988). Raft association may favour the
preservation of the active receptor conformation, and clustering
of individual rafts could account for the observed receptor
aggregation, helping to potentiate signalling by concentrating
downstream components, as has been shown for other signalling
cascades. It is also conceivable that IR is constitutively associ-
ated with rafts but has only moderate affinity for rafts in vivo that
can be disrupted by non-ionic detergents, as suggested for the
T-cell receptor (Janes et al., 1999). In this scenario, IR activation
could involve aggregation and stabilization of rafts already
harbouring the receptor.
METHODS
Cyclodextrin treatments. To deplete cholesterol, HuH7 cells
(Nakabayashi et al., 1982) were incubated with 10 mM methyl-
β-cyclodextrin for 5 min at 37°C. Insulin (1.0 IU/ml) was added
to the indicated samples, and the incubation continued for
10 min. Cells were then lysed for western blotting or gradient
fractionation. To measure cholesterol efflux, cells were labelled
overnight with 0.05 µCi/ml [
14
C]cholesterol in growth medium
and incubated in the presence of cyclodextrin as above. Radio-
activity from cells and media was measured by liquid-scintilla-
tion counting. To replete cholesterol, depleted cells were
incubated for 2 h with methyl-β-cyclodextrin/cholesterol
complex (molar ratio 8:1).
Glycosphingolipid clustering. Cells were incubated at 12°C for
1 h in the presence of 20 µg/ml anti-GM2 [mouse IgM against
human GM2 (Karlsson et al., 1990)] or irrelevant antibody (anti-
LDL receptor antibody C7 or mouse IgM MOPC 104C), washed
and incubated for 1 h at 12°C in the presence of secondary anti-
bodies. After insulin stimulation at 37°C, cells were lysed for
western blotting or gradient fractionation. Cells on cover slips
were fixed for 2 h in 4% PFA at room temperature after incub-
ation with the secondary antibodies. To visualize GM2 staining
without patching, staining with the secondary antibody was
done after fixation. To examine the distribution of occupied IR in
patched and non-patched cells, FITC-labelled insulin (30 ng/µl)
was added for 5 min at 12°C before fixation.
Western blotting, flotation gradients. For western blotting, cells
were lysed in buffer containing 1% SDS, 10 mM TrisHCl,
pH 7.5, phosphatase and protease inhibitors and 20 µg of
protein resolved by SDSPAGE. For western blotting with anti-
PY antibody, filters were blocked with 3% BSA; otherwise
5% milk was used. The signal visualized by enhanced chemi-
luminiscence detection was quantified by normalizing the
intensity of the anti-PY band with the intensity of the IR band in
the same blot. The association of IR with DRMs was studied by
lysing the cells in 0.1% Triton X-100 on ice and fractionating in
TritonOptiprep gradients according to Heino et al. (2000).
Proteins TCA precipitated from the fractions were analysed by
western blotting.
Supplementary data. Supplementary data are available at
EMBO reports Online.
ACKNOWLEDGEMENTS
We thank Antti Virkamäki for valuable comments. This work
was financially supported by the Sigrid Juselius Foundation,
Jenny and Antti Wihuri Foundation, Swedish Medical Research
council and the Academy of Finland.
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DOI: 10.1093/embo-reports/kvf010
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  • Source
    • "These results indicate a possible mechanism for the lower tumorigenic capacity of Ir-KD cells; however, it is important to note that CD24 (in the Ir-KD/CD24 C cells) did not rescue the cells' response to insulin in vitro and, on the other hand, insulin treatment for up to 3 weeks had no effect on in vitro expression of CD24 (data not shown). Lipid rafts are plasma membrane domains that are known to contain many signaling proteins including IR (Vainio et al. 2002). Results from many studies have indicated that lipid rafts are critical for proper insulin activation of both the PI3K/Akt signaling pathway and the MAPK pathway (Bickel 2002). "
    [Show abstract] [Hide abstract] ABSTRACT: Accumulating evidence from clinical trials indicates that specific targeting of the IGF1 receptor (IGF1R) is not efficient as an anti-breast cancer treatment. One possible reason is that the mitogenic signals from the insulin receptor (IR) can be processed independently or as compensation to inhibition of the IGF1R. In this study, we highlight the role of the IR in mediating breast tumor progression in both WT mice and a hyperinsulinemic MKR mouse model by induction of Ir (Insr) or Igf1r knockdown (KD) in the mammary carcinoma Mvt-1 cell line. By using the specific IR antagonist-S961, we demonstrated that Igf1r-KD induces elevated responses by the IR to IGF1. On the other hand, Ir-KD cells generated significantly smaller tumors in the mammary fat pads of both WT and MKR mice, as opposed to control cells, whereas the Igf1r-KD cells did not. The tumorigenic effects of insulin on the Mvt-1 cells were also demonstrated using microarray analysis, which indicates alteration of genes and signaling pathways involved in proliferation, the cell cycle, and apoptosis following insulin stimulation. In addition, the correlation between IR and the potential prognostic marker for aggressive breast cancer, CD24, was examined in the Ir-KD cells. Fluorescence-activated cell sorting (FACS) analysis revealed more than 60% reduction in CD24 expression in the Ir-KD cells when compared with the control cells. Our results also indicate that CD24-expressing cells can restore, at least in part, the tumorigenic capacity of Ir-KD cells. Taken together, our results highlight the mitogenic role of the IR in mammary tumor progression with a direct link to CD24 expression. © 2015 Society for Endocrinology.
    Full-text · Article · Apr 2015 · Endocrine Related Cancer
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    • "Therefore, it is tempting to speculate that PHBs binding to TRPM6 promotes the formation of a macromolecular regulatory complex in the lipid rafts of the plasma membrane. In this perspective, it is interesting to note that the insulin-induced signaling pathway becomes more active when the insulin receptor is expressed in lipid rafts323334. In addition to the inhibitory PHB2-TRPM6 interaction, other TRP channels are modulated by members of the SPFH protein family [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Magnesium (Mg 2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg 2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg 2+ (re)ab-sorption in kidney and intestine. The apical entry of Mg 2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses , it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by *2 fold. The stimulatory effects were dependent on the presence of the alpha-ki-nase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.
    Full-text · Article · Mar 2015 · PLoS ONE
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    • "The IR has also been observed in caveolin-enriched hepatic plasma membrane microdomains that rapidly associate with the actin cytoskeleton in response to insulin [58] . IR autophosphorylation is thought to release a constraint that maintains the IR on the microvilli and allows free, mobile insulin-bound IR to reach competent membrane environments [59,60]. The link with the actin network appears to be a key component of IR traffic because ANXA2 is also linked with the CEACAM1/b-catenin complexes (Fig. 5) that were shown to be involved in glucose metabolism and IR regulation [61,62]. "
    [Show abstract] [Hide abstract] ABSTRACT: Insulin receptor (IR) endocytosis requires a remodelling of the actin cytoskeleton. We show here that ANXA2 is SUMOylated at the K10 located in a non-consensus SUMOylation motif in the N-terminal domain. The Y24F mutation decreased the SUMOylation signal, whereas insulin stimulation increased ANXA2 SUMOylation. A survey of protein SUMOylation in hepatic Golgi/endosome (G/E) fractions after insulin injections revealed the presence of a SUMOylation pattern and confirmed the SUMOylation of ANXA2. The construction of an IR/ANXA2/SUMO network (IRASGEN) in the G/E context reveals the presence of interacting nodes whereby SUMO1 connects ANXA2 to actin and microtubule-mediated changes in membrane topology. Heritable variants associated with type 2 diabetes represent 41% of the IRASGEN thus pointing out the physio-pathological importance of this subnetwork. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · FEBS letters
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