EMBO reports vol. 3 | no. 1 | 2002 99
Insulin signalling without caveolae
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
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.
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 [
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 Tris–HCl,
pH 7.5, phosphatase and protease inhibitors and 20 µg of
protein resolved by SDS–PAGE. 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
Triton–Optiprep gradients according to Heino et al. (2000).
Proteins TCA precipitated from the fractions were analysed by
Supplementary data. Supplementary data are available at
EMBO reports Online.
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.
Calvo, M., Tebar, F., Lopez-Iglesias, C. and Enrich, C. (2001) Morphologic
and functional characterization of caveolae in rat liver hepatocytes.
Hepatology, 33, 1259–1269.
Carpentier, J.L., Fehlmann, M., Van Obberghen, E., Gorden, P. and Orci, L.
(1985) Redistribution of 125I-insulin on the surface of rat hepatocytes as
a function of dissociation time. Diabetes, 34, 1002–1007.
Carpentier, J.L., Paccaud, J.P., Gorden, P., Rutter, W.J. and Orci, L. (1992)
Insulin-induced surface redistribution regulates internalization of the
insulin receptor and requires its autophosphorylation. Proc. Natl Acad.
Sci. USA, 89,162–166.
Couet, J., Sargiacomo, M. and Lisanti, M.P. (1997) Interaction of a receptor
tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively
regulates tyrosine and serine/threonine kinase activities. J. Biol. Chem.,
Fielding, C.J. and Fielding, P.E. (2000) Cholesterol and caveolae: structural
and functional relationships. Biochim. Biophys. Acta, 1529, 210–222.
Gustavsson, J. et al. (1999) Localization of the insulin receptor in caveolae of
adipocyte plasma membrane. FASEB J., 13, 1961–1971.
Harder, T., Scheiffele, P., Verkade, P. and Simons, K. (1998) Lipid domain
structure of the plasma membrane revealed by patching of membrane
components. J. Cell Biol., 141,929–942.
Hedo, J.A., Collier, E. and Watkinson, A. (1987) Myristyl and palmityl
acylation of the insulin receptor. J. Biol. Chem., 262,954–957.
Heffetz, D. and Zick, Y. (1986) Receptor aggregation is necessary for
activation of the soluble insulin receptor kinase. J. Biol. Chem., 261, 889–894.
Heino, S., Lusa, S., Somerharju, P., Ehnholm, C., Olkkonen, V.M. and
Ikonen, E. (2000) Dissecting the role of the Golgi complex and lipid rafts
in biosynthetic transport of cholesterol to the cell surface. Proc. Natl
Acad. Sci. USA, 97,8375–8380.
Drbal, K. (1998) Signal transduction in leucocytes via GPI-anchored
proteins: an experimental artefact or an aspect of immunoreceptor
function? Immunol. Lett., 63,63–73.
Hubbard, S.R., Wei, L., Ellis, L. and Hendrickson, W.A. (1994) Crystal
structure of the tyrosine kinase domain of the human insulin receptor.
Janes, P.W., Ley, S.C. and Magee, A.I. (1999) Aggregation of lipid rafts
accompanies signalling via the T cell antigen receptor. J. Cell Biol., 147,