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J Biol Chem, Vol. 274, Issue 49, 34981-34992, December 3, 1999
,§,
From the Departments of Biophysics and Biochemistry, Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Insulin binding to the insulin receptor
initiates a cascade of cellular events that are responsible for
regulating cell metabolism, proliferation, and growth. We have
investigated the structure of the purified, functionally active, human
insulin receptor using negative stain and cryo-electron microscopy.
Visualization of the detergent-solubilized and vesicle-reconstituted
receptor shows the The insulin receptor is a well known transmembrane protein that
has been the focus of extensive scientific study for over 3 decades. It
is through this receptor that the peptide hormone insulin regulates a
multitude of cellular processes including glucose transport and
metabolism, fatty acid metabolism, DNA and protein synthesis, amino
acid transport, and mitogenesis. Insulin binding to the extracellular
domain of the insulin receptor results in receptor autophosphorylation
and activation (reviewed in Ref. 1). The phosphorylated insulin
receptor activates many intracellular signaling pathways (reviewed in
Ref. 2) through the insulin receptor substrate
(IRS)1 family members and
phosphatidylinositol 3'-kinase, which is upstream of protein kinase C
and protein kinase B/Akt enzymes (3). How these kinases are
biochemically connected to ultimate targets, such as glucose
transporters, remains obscure. Despite the enormous amount of
information regarding the insulin signaling pathway, information
detailing structural aspects of the insulin receptor itself is limited.
The insulin receptor is a member of the receptor tyrosine kinase family
(4). This receptor shares considerable sequence similarity and
structural characteristics with the insulin like growth factor-1
(IGF-1) receptor (4). The insulin receptor is a glycosylated
heterotetrameric protein composed of two A subdomain organization of the insulin receptor ectodomain has been
proposed based on sequence comparison with the epidermal growth factor
receptor (11), growth hormone receptor and tenascin (12), and the tumor
necrosis factor receptor-1 (13). The ectodomain is composed of two
large homologous domains, L1 (residues 1-119) and L2 (residues
313-428), separated by several cysteine-rich domains (residues
155-312) (11). Secondary structural predictions suggest that each
large homologous domain is composed of an Segments of the cytoplasmic domain of the insulin receptor have also
been crystallized. A peptide fragment of the insulin receptor
juxtamembrane region has been crystallized complexed with the IRS-1
phosphotyrosine binding domain (16). The resulting structure
demonstrated the importance of specific structural features, including
a key phosphotyrosine residue, in this interaction (16). The tyrosine
kinase domain of the insulin receptor has also been crystallized in
both the dephosphorylated (17) and phosphorylated (18) forms. The
crystal structure of the kinase region enabled identification of key
residues involved in the specificity of the family of tyrosine kinases.
The phosphorylated form of the kinase region demonstrated the
conformational changes that take place in the activated receptor
exposing the nucleotide binding site and positioning the catalytic loop
for substrate interactions. These structures cannot, however, be used
to address the importance of interactions between the two Electron microscopic studies allow one to study the holoreceptor and
the domain organization that so far cannot be captured in a crystalline
state. Negative stain electron microscopic studies of the purified
placental Cryo-electron microscopy enables visualization of a protein in a fully
hydrated state, free of heavy metals and the associated artifacts.
Macromolecular complexes are preserved in a non-crystalline, or
vitreous ice layer and visualized under low electron dose conditions to
minimize radiation damage (21). The relative background noise is
higher, and the contrast between protein and background is lower than
in stain; however, the structural detail of a biological sample is more
accurately preserved. We have applied the technique of cryo-electron
microscopy to the study of insulin receptor structure and visualized
the insulin receptor in both the detergent-solubilized and
vesicle-reconstituted forms. Using specific labeling techniques, we
have also been able to identify distinct subdomains of the heterotetrameric complex under cryo conditions. These studies demonstrate the contribution that electron microscopy can make to
understanding the entirety of a complex and structurally challenging protein such as the insulin receptor.
Insulin Receptor Purification
Purification Protocol--
Fifteen dishes (500 cm2)
(Vanguard, Neptune, NJ) of NIH-3T3 cells overexpressing the human
insulin receptor cDNA (1502 cells, kindly provided by Dr. Simeon
Taylor, National Institutes of Health, Bethesda, MD) were grown to
confluence in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) with 10% heat-inactivated fetal bovine sera (Life Technologies,
Inc.) and 0.1 g/liter Geneticin (Life Technologies, Inc.). Cells were
washed twice with 50 ml of phosphate-buffered saline containing a
protease inhibitor mixture consisting of 10 µM leupeptin
(Roche Molecular Biochemicals), 0.1 TIU/ml aprotinin (American
Bioanalytical, Natick, MA), 1 µM pepstatin (Sigma), 1 mM o-phenanthroline (Sigma), 1 mM
phenylmethylsulfonyl fluoride (American Bioanalytical), 25 mM benzamidine·HCl (Sigma), and 5 mM EDTA.
Cells were then lysed with 50 ml of 10 mM HEPES also
containing the protease inhibitor mixture. This first lysis solution
was poured off and discarded before a second 50 ml of 10 mM
HEPES with the protease inhibitor mixture was added. While in the
second lysis buffer, the remaining adherent cellular components were
scraped from the plates and poured into 250-ml centrifuge tubes on ice.
The membranes in lysis buffer were centrifuged in a GSA rotor for
1 h at 21,520 × g at 4 °C. The supernatant was discarded, and the pellet was stored (for no longer than 1 month) at
Frozen membranes were thawed on ice. The membranes were resuspended in
30 mM HEPES with the protease inhibitor mixture. The resuspended membranes were transferred to a 50-ml conical tube. 10%
Triton X-100 (Roche Molecular Biochemicals) was added to a final
concentration of 2%, and the membranes were incubated with gentle
agitation at 4 °C for 1 h. The solubilized membranes were centrifuged in a Ti70 rotor for 75 min at 148,600 × g
at 4 °C. The supernatant was collected and filtered through several
0.45-µm syringe filters (Nalgene, Rochester, NY).
The filtered supernatant was loaded onto a 1.6-liter bed volume
Sephacryl 400 (Amersham Pharmacia Biotech) column using a 50-ml maximum
volume Superloop (Amersham Pharmacia Biotech). The column was
controlled by fast pressure liquid chromatography at a rate of 1.33 ml/min, and 12-ml fractions were collected between 650 and 1650 ml. The
first 80 fractions were assayed for protein amount using the
Bradford-based Bio-Rad protein assay. An insulin binding assay was
performed on 10 µl from every odd fraction in the region immediately
preceding the protein peak and including the first few fractions of the
protein peak itself.
The 125I insulin solution for the insulin binding assay
(adapted from Refs. 22 and 23) consisted of 125I insulin
(NEN Life Science Products) in 3 ml of 1 mg/ml solution of BSA (Sigma)
in 30 mM HEPES until the cpm of 90 µl was between 15,000 and 20,000 cpm. 90 µl of 125I insulin solution was added
to 10 µl from each chosen fraction, and incubated at room temperature
for 45 min. 250 µl of 1.25 mg/ml
Pooled Sephacryl 400 fractions were placed over a 5-ml bed volume wheat
germ agglutinin (EY Laboratories, San Mateo, CA) column (24)
equilibrated with 500 ml of 30 mM HEPES, 0.1% Triton
X-100, 0.02% NaN3, and protease inhibitor mixture using a
peristaltic pump until at least three times the applied volume had been
passed over the wheat germ agglutinin column. The column was then
washed with a minimum volume of 500 ml of 30 mM HEPES,
0.1% Triton X-100, 0.02% NaN3, and protease inhibitor
mixture. The protein was eluted with 40 ml of 0.3 M
N-acetyl glucosamine (Sigma) in 20 mM Tris with
0.1% Triton X-100 without protease inhibitors over a period of 1-2 h.
The entire wheat germ agglutinin column eluant was loaded onto a 1-ml
MonoQ column (HR 5/5; Amersham Pharmacia Biotech), equilibrated with 20 mM Tris and 0.1% Triton X-100 using a Superloop, and
controlled by fast pressure liquid chromatography at a rate of 1 ml/min. The column was then washed with 20 ml of 20 mM Tris
and 0.1% Triton X-100, and eluted with a 120-ml linear gradient from
0% to 50% of 1 M NaCl. 1-ml fractions were collected
through the entire gradient and assayed for protein content. The
insulin binding assay was carried out as described above, on 2 µl from the fractions that constitute the protein peak. Fractions
from the MonoQ column that centered around the second insulin binding
peak, corresponding to
The pooled MonoQ fractions were diluted 3-fold with 20 mM
Tris, 0.1% Triton X-100 before being continuously loaded onto a 500-µl bed volume DEAE column that had been equilibrated with 20 mM Tris and 0.1% Triton X-100. After at least three times
the applied volume was allowed to pass over the column, the DEAE column was washed with 50 ml of 20 mM Tris and 0.1% Triton X-100.
The column was then eluted by layering 500 µl of 20 mM
Tris, 0.1% Triton X-100, 0.5 M NaCl onto the column and
allowing it to incubate for approximately 5 min before collecting the
eluant. This was repeated a total of six times to ensure maximal
protein recovery. 2 µl from each of the six elutions were assayed for
protein amount, and the first three fractions were pooled for the final
gel filtration step.
The Superose 6 (Pharmacia) column consisted of two 100-ml bed volume
columns connected in series and was equilibrated with 600 ml of 30 mM HEPES, 0.1% Triton X-100, 0.02% NaN3, and
150 mM NaCl. The pooled fractions from the DEAE column were
loaded onto the Superose 6 column using a 2-ml sample loop, and
controlled by fast pressure liquid chromatography at a rate of 0.33 ml/min. 1-ml fractions were collected between 60 and 200 ml. As
described above, 20 µl of every odd fraction was assayed for protein
amount, and 10 µl of the fractions surrounding and including the
protein peak were assayed for insulin binding activity. The fractions containing the highest amount of both insulin binding activity and
protein amount were pooled.
The pooled Superose 6 fractions were diluted 3-fold with 20 mM Tris and 0.1% Triton X-100, and loaded onto a new DEAE
column (100 µl bed volume) for concentration and detergent exchange. The diluted fractions were continuously loaded onto the DEAE column equilibrated with 20 mM Tris, and 0.1% Triton X-100 until
at least three times the diluted volume had passed over the column. The column was washed with 5 ml of 20 mM Tris, and 0.6%
SDS-PAGE--
The relative protein composition and insulin
receptor purity at each step of the purification was determined by
SDS-PAGE. The protein amounts from each step were loaded to show
relatively constant amounts of insulin receptor through each stage of
the purification procedure. Samples were run on a 3-10% gradient gel under non-reducing conditions using the Laemmli buffer system (25). The
gel was immediately transferred to a fixative solution (40% methanol
and 10% acetic acid) and silver stained (Bio-Rad).
Western Blotting--
A second gel was run under the same
conditions as above, using the same samples, and subjected to Western
blotting. After the gel was electrophoretically transferred to
Immobilon-P (Millipore, Bedford, MA) by wet blotting (1250 total
milliamps), the membrane was blocked in a 10% milk in 30 mM HEPES, 150 mM NaCl, 0.01% NaN3 solution for 2 h at room temperature and rinsed twice with PBST. The primary antibody, polyclonal anti-insulin receptor Autophosphorylation Assay--
30 mM HEPES was added
to 0.5 µg of purified receptor to a final volume of 40 µl. 5 µl
of insulin (10 Labeling of the Insulin Receptor
Streptavidin-Nanogold--
30 µg of purified insulin receptor
in 20 mM Tris and 0.1% Triton X-100 was incubated with
10
300 µl of wheat germ agglutinin-agarose beads (50% beads v/v)
equilibrated in 30 mM HEPES, 0.1% Triton X-100, 0.02%
NaN3, and the protease inhibitor mixture (see purification
protocol) were added to the cross-linked BBpa insulin·receptor
complex and incubated with gentle agitation overnight at 4 °C. The
beads were settled with a 3-s pulse in a benchtop microcentrifuge at
4 °C, and the supernatant containing unbound streptavidin-Nanogold
and uncross-linked BBpa insulin was removed. The wheat germ
agglutinin-agarose beads were washed three times with 500 µl of 20 mM Tris and 0.1% Triton X-100 without protease inhibitors
for 10 min. At each wash, the beads were settled with a 3-s pulse in a
benchtop microcentrifuge at 4 °C and the supernatant was removed.
The wheat germ agglutinin-agarose was eluted by incubating the beads
for 2 h with 150 µl of 0.6 M
N-acetylglucosamine in 20 mM Tris, 0.1% Triton
X-100. The beads were settled, and the supernatant containing the
gold-labeled insulin receptor was removed. This was followed by two
more elutions of 150 µl each and incubation periods of 30 min. The
three elutions were pooled.
200 µl of Q-Sepharose beads (Amersham Pharmacia Biotech) (50% v/v)
equilibrated in 20 mM Tris, 0.1% Triton X-100 were added to the wheat germ elution pools and allowed to incubate overnight at
4 °C with gentle agitation. The beads were settled using pulse centrifugation at 4 °C, and the supernatant was removed. The
Q-Sepharose beads were washed three times with 500 µl of 20 mM Tris and 0.6%
The gold-labeled insulin receptor was concentrated, and the salt was
removed using a Microcon-50 microconcentrator (Amicon) blocked in 1%
milk overnight. 400 µl of the Q-Sepharose pools were added to the
Microcon-50, diluted to 500 µl, and centrifuged at 12,700 × g for 10 min. 200 µl of 20 mM Tris, 0.6%
1-Biotinamido-4-4[4'-(maleimidomethyl) cyclohexane
carboxamido] Butane (Biotin-BMCC)--
Biotin-BMCC (Pierce) was
suspended in Me2SO (Sigma) to a final concentration of 10 mM. 2 µg of purified insulin receptor was brought to a
total volume of 18 µl with 30 mM HEPES, 0.1% Triton X-100, 0.02% NaN3, pH 7.4. Biotin-BMCC was added to a
final concentration of 1 mM and incubated at room
temperature for 1 h. Samples were split into two tubes, each
containing 9 µl of the reaction mix. To one of these tubes, sample
buffer containing DTT was added to a final concentration of 150 mM DTT, heated at 100 °C for 5 min, and allowed to cool.
Samples were run on a 7% polyacrylamide mini-gel using the Laemmli
buffer system and electrophoretically transferred to Immobilon-P by wet
blotting. The membrane was blocked for 2 h at room temperature in
a 10% milk solution in 30 mM HEPES, 150 mM
NaCl, and 0.02% NaN3. The membrane was incubated with
streptavidin·HRP (Amersham Pharmacia Biotech) for 1 h at
37 °C and washed three times for 15 min with PBST. The signal was
detected by using an enhanced chemiluminescence system and exposing the
membrane to film. The peroxidase enzyme was inactivated by incubating
the membrane for 30 min at room temperature in 15%
H2O2 in phosphate-buffered saline, followed by
three 10-min washes in PBST. The insulin receptor Monomaleimide-Nanogold--
9.2 µg of purified insulin
receptor was dialyzed using a dialysis membrane molecular weight
cut-off of 50,000 (Spectrum, Houston, TX) at 4 °C against 100 ml of
30 mM HEPES, 0.6% Reconstitution
Formation of Large Unilamellar Vesicles--
The concentration
of egg phosphatidylcholine (eggPC) (Avanti Polar Lipids Inc.,
Alabaster, AL) in choloroform was calculated by dry weight using a Cahn
Balance. The volume corresponding to 1 mg of eggPC was added to a 25-ml
round-bottomed flask, and the choloroform was evaporated using a
Rotovac. Approximately 5 ml of 2:1 chloroform:methanol were added to
the round bottom flask, the lipid was redissolved, and the solvent was
then evaporated using a Rotovac. The addition and evaporation of 5 ml
of 2:1 chloroform:methanol was repeated two more times until a thin,
even lipid film lined the inside of the flask. The dried eggPC film was
then lyophilized, at room temperature, in the dark, overnight.
The lyophilized lipid film was resuspended in 500 µl of 30 mM HEPES, 50 mM NaCl, and 0.01%
NaN3 to a final concentration of 2 mg/ml. After the
solution appeared opaque, it was transferred to a 1.5-ml Eppendorf tube
for the freeze/thaw procedure. The vesicle solution was repeatedly
placed in a liquid N2 bath for 30 s and then
immediately placed in a 50-60 °C water bath until thawed for a
total of six times. The resultant particulate vesicle solution was
extruded through two 0.1-µm filters at room temperature for a total
of 10 times to produce large unilamellar vesicles (27).
Reconstitution Dialysis--
4 µg of purified
Electron Microscopy
Negative Stain Electron Microscopy--
The samples were diluted
to the appropriate concentration using buffer that contained the lowest
possible amounts of salt and detergent. Control vesicles and
reconstitutions were diluted to a lipid concentration of 0.1 mg/ml with
30 mM HEPES, 25 mM NaCl, and 0.01%
NaN3. Detergent-solubilized receptor was diluted to a
protein concentration of 5 µg/ml with 30 mM HEPES, 50 mM NaCl, 0.6%
Negative stained samples were visualized in a Philips CM12 transmission
electron microscope using a room temperature, single-tilt holder. The
high tension setting used was 120 kV, the condenser aperture was 100 µm, and the objective aperture was 50 µm. The camera was set for
0.701 s in manual mode, and the beam intensity was adjusted to a meter
reading of 1.47 s. Micrographs were recorded at 300-500 nm
underfocus. All data was collected on Kodak Electron Image Film SO-163
(magnification, ×75,000), under low dose conditions to minimize
irradiation and sample damage giving a total electron dose of less than
30 e/Å2. Negatives were developed in concentrated D19 for
12 min.
Cryo-electron Microscopy--
Holey grids were prepared based on
the method by Harris (28). Grids were etched in 100% methanol and
dried. The dried grids were immediately carbon-coated in a Denton
evaporator with half of a carbon rod tip (0.040-inch diameter,
1/16-inch length) (Ladd Research Industries Inc., Burlington, VT).
Immediately prior to use, holey grids used for the
detergent-solubilized receptor were glow-discharged in
n-amylamine at 0.08 mbar twice for 29 s at 150 V
corresponding to 2.5 mA. Holey grids used for vesicles and reconstitutions were glow-discharged in air at 0.08 mbar three times
for 29 s at 150 V corresponding to 2.5 mA.
6 µl of sample (1 mg/ml for control vesicles, 300 µg/ml for
detergent-solubilized receptor, 0.5 mg/ml for reconstitutions) was
placed on a holey grid and allowed to incubate for approximately 30 s. Excess liquid was blotted from the back side of the grid for
3 s using Whatman no. 1 paper. The grid was immediately plunged, using an N2-gas driven plunging apparatus, into liquid
ethane-cooled in a liquid N2 bath. The grid was maintained
in liquid N2 until visualized. The entire plunging process
was carried out at room temperature in a humidified chamber.
Samples prepared for cryo-electron microscopy were transferred to a
Gatan cryo-holder (Gatan, Inc., Pittsburgh, PA) using a Gatan
cryotransfer station and maintained at a temperature less than
Image Analysis--
Negatives were digitized using an Eikonix
Imaging System at a 37.5 µm raster and a camera height of 55.7 cm,
corresponding to 5 Å/pixel. Selected regions were scanned using a
Nikon 60-mm lens at an f-stop of 5.6. The Eikonix was controlled by the
xscantool software package and a SUN Microsystems workstation, which
generated the scanned images in Sun raster format. The scanned images
were transferred to an open VMS alpha workstation, where they were converted into SPIDER format. Individual particle selection and image
manipulation was done using the SPIDER software package (29) and the
associated viewer WEB (29). Contrast was corrected by multiplying the
density value of each pixel by Insulin Receptor Purification
Insulin receptors were purified from NIH-3T3 cells overexpressing
the human insulin receptor cDNA. Isolated cell membranes were
solubilized in Triton X-100, and the resultant protein solution was
subjected to conventional chromatographic separation including gel
filtration, affinity chromatography, and ion exchange chromatography. The presence of the insulin receptor was monitored throughout the
purification using protein and insulin binding assays (data not shown).
The final product from the purification is a single protein of
approximately 350 kDa under non-reducing conditions (Fig.
1A, lane
6) confirmed as the heterotetrameric insulin receptor by
Western blotting (Fig. 1B, lane 6).
Despite the absence of reducing agent, a distinct band of approximately
200 kDa was often present during the purification, and this was
identified as 
2
2
heterotetrameric insulin receptor to be a three-armed pinwheel-like
complex that exhibits considerable variability among individual
receptors. The -subunit of the receptor was labeled with an insulin
analogue·streptavidin gold conjugate, which facilitated the
identification of the receptor arm responsible for insulin binding. The
gold label was localized to the tip of a single receptor arm of the
three-armed complex. The -subunit of the insulin receptor was
labeled with a maleimide-gold conjugate, which allowed orientation of
the receptor complex in the membrane bilayer. The model derived from
electron microscopic studies displays a "Y"-like morphology representing the predominant species identified in the reconstituted receptor images. The insulin receptor dimensions are approximately 12.2 nm by 20.0 nm, extending 9.7 nm above the membrane surface. The
-subunit-containing arm is approximately 13.9 nm, and each -subunit-containing arm is 8.6 nm in length. The model presented is
the first description of the insulin receptor visualized in a fully
hydrated state using cryo-electron microscopy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits and two
-subunits. Each -subunit is covalently linked to an -subunit
by class II disulfide bonds to form an heterodimer (5). Two
heterodimers are covalently linked through the -subunits by
class I disulfide bonds to form 22
heterotetramers (5). The -subunits of the insulin receptor are
entirely extracellular and contain the insulin binding site (reviewed
in Ref. 1). Each -subunit consists of an extracellular domain, a
transmembrane domain, and an intracellular domain. The intracellular
domain of the -subunit contains the kinase activity of the insulin
receptor, and this segment can be further subdivided into a
juxtamembrane region, a kinase region, and a C-terminal region. Binding
of insulin to the -subunit of the insulin receptor results in a
conformational change that is coincident with the autophosphorylation
of the -subunit of the receptor (6). Autophosphorylation is an
intramolecular process (7, 8) that occurs in trans (9, 10)
such that one -subunit phosphorylates the tyrosine residues on the
other -subunit in the heterotetramer.
-helix, -strand, turn,
-strand motif (11). The cysteine-rich regions of the insulin
receptor may adopt one or two loop configurations similar to those
identified in the tumor necrosis factor receptor-1 (13). The
carboxyl-terminal segment of the insulin receptor extracellular domain
contains two potential fibronectin type III repeats, each consisting of
a seven-stranded -sheet structure. This structural information
relies heavily on sequence based predictions. A segment of the IGF-1
receptor extracellular domain containing the L1-cystine-rich-L2 regions
has recently been crystallized (14) and solved (15). The crystal
structure exhibits this region of the monomeric -subunit fragment as
a relatively extended segment (40 × 48 × 105 Å) (15). This
fragment is the first successfully crystallized extracellular segment
of the insulin receptor class of proteins, and the information obtained
begins to provide insight into the extracellular organization of this
class of proteins.
-subunits
of the heterotetrameric complex, nor can they address the role that
insulin binding plays in receptor activation.
22 heterotetrameric insulin
receptor have identified "Y"-like and "T"-like conformations in
the detergent-solubilized state (19). These studies are supported by
images of the extracellular domain of the receptor identifying short
Y-like and "V"-like structures (12). Images of the reconstituted
insulin receptor visualized using negative stain electron microscopy
also show Y-like, V-like, T-like, and stalklike projections from the
vesicle surface (20). All of the microscopic studies to date are
limited by the use of heavy metals for preservation and contrast. These
heavy metals potentially distort structural detail, in addition to
requiring a certain degree of sample drying.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C.
-globulins (Sigma) in 30 mM NaPO4, pH 7.4, and 250 µl of 25% polyethylene glycol (Sigma) in 30 mM NaPO4, pH
7.4, were added and incubated at 4 °C for 45 min. The samples were
centrifuged at 15,000 × g at 4 °C for 20 min. The
supernatant was aspirated, and the remaining pellet was counted in a
-counter. Results were expressed as the percentage of total insulin
binding (cpm in the pellet/total cpm in 90 µl of 125I BSA
solution) × 100 and graphed along with the protein profile. Fraction corresponding to the insulin binding peak were pooled and
applied to the next column.
22 holoreceptor
(data not shown), were pooled. The first insulin binding peak,
identified as half-receptor, was pooled independently and
further purified in a procedure identical to that for the
22 holoreceptor. The pooled fractions
were loaded onto a DEAE (Fluka, Milwaukee, WI) column for concentration.
-octyl glucoside (Pfanstiehl Laboratories, Waukegan, IL) and eluted
by layering 100 µl of 20 mM Tris, 0.6% -octyl
glucoside, 10% glycerol, and 0.5 M NaCl onto the column
and allowing it to incubate for approximately 10 min before collecting
the eluant. The elution procedure was repeated a total of six times to
ensure maximal protein recovery. 1 µl of each fraction was assayed
for protein amount, and those containing more than 200 µg/ml protein
were aliquoted for storage at 80 °C. Normal protein yields were
approximately 200 µg/purification.
-subunit antibody (R1064) (26) in 2% BSA in PBST, was incubated on the membrane
at 37 °C for 2 h. The membrane was washed three times for 10 min at 37 °C before incubation with the enzyme-linked conjugate, Protein A/G HRP (horseradish peroxidase) (Pierce). The Protein A/G HRP
was diluted 10,000 times in 2% BSA in PBST and incubated on the
membrane at 37 °C for 2 h after which the membrane was washed
again as above. The signal was detected by using an enhanced chemiluminescence system (Pierce) and exposing the membrane to film (Sigma).
6 M initial concentration)
(Sigma) or 5 µl of 30 mM HEPES was added, and samples
were incubated at room temperature for 45 min. 5 µl of a 10× ATP
mixture (100 mM MgCl2, 80 mM
MnCl2, 500 µM ATP) was added to all of the
samples, which were then incubated at room temperature for 20 min. The
reaction was stopped by adding 10 µl of 400 mM EDTA.
Samples were electrophoretically separated and transferred to Immobilon
as described above. After blocking, the first antibody (4G10
anti-phosphotyrosine) (Upstate Biotechnology, Lake Placid, NY) was
incubated in 4% BSA in PBST at 37 °C for 2 h. The membrane was
washed three times in PBST for 10 min at 37 °C before incubation
with the secondary antibody anti-mouse IgG·HRP (Sigma) in 2% BSA in
PBST at 37 °C for 2 h. The signal was detected as above. The
membrane was then rinsed for 15 min in PBST before being incubated with
the anti-insulin receptor -subunit antibody R1064 (see above).
6 M B25-L-benzoylphenylalanine,
B29-biotinyl insulin (BBpa insulin) (26) in a total volume of 100 µl
overnight, in the dark, at 4 °C. The BBpa insulin·receptor
solution was exposed to 3 amps of UV light at a wavelength of 345 nm
for 1 h on ice. Streptavidin-Nanogold (Nanoprobes, Stony Brook,
NY) was added to a 5-fold molar excess over BBpa insulin and incubated
at 4 °C for 1 h.
-octyl glucoside for 15 min. At each
wash, the beads were settled with a 3-s pulse in a benchtop
microcentrifuge at 4 °C, and the supernatant was removed. The
Q-Sepharose was eluted by incubating the beads with 100 µl of 20 mM Tris, 0.75% -octyl glucoside, and 1 M
NaCl for 30 min. The beads were settled, and the supernatant was
removed. The beads were eluted two additional times by incubating 100 µl of 20 mM Tris, 0.6% -octyl glucoside, and 1 M NaCl for 15 min before settling the beads and removing
the supernatant. The three elutions were pooled and concentrated.
-octyl glucoside, 100 mM NaCl, and
0.01%NaN3 were added, and the Microcon was spun again for
13 min. 50 µl of 20 mM Tris, 0.6% -octyl glucoside, 100 mM NaCl, and 0.01% NaN3 was added to the
Microcon, which was then inverted and pulsed. Gold-labeled insulin
receptor was visualized by electron microscopy.
-subunit was
incubated with a polyclonal anti- subunit peptide antibody (R1064)
in 2% BSA in PBST for 2 h at 37 °C and washed three times for
10 min at 37 °C in PBST. Protein A/G HRP (diluted 1:10,000 in 2%
BSA in PBST) was incubated with the membrane for 1 h at 37 °C.
The membrane was washed three times for 10 min at 37 °C in PBST, and
the signal was detected as described above. The -subunit was
identified by incubating the same membrane with an anti--subunit
antibody (Upstate Biotechnology, Inc.) in 2% BSA in PBST overnight at
4 °C, washing three times for 10 min with PBST, followed by
incubation with goat anti-rabbit IgG·HRP in 2% BSA in PBST for
1 h at 37 °C. The blot was washed, and the signal was detected
as previously mentioned.
-octyl glucoside, and 50 mM NaCl, without NaN3, for 1.5 h followed
by dialysis against 350 ml of fresh buffer for 4 h. Six nanomoles
of monomaleimide-Nanogold was suspended in 20 µl of isopropanol and
brought to a final volume of 200 µl with distilled water, as
suggested by Nanoprobes, immediately prior to use. Assuming that the
cysteine of each subunit in the 22
heterotetramer was available for labeling, a 10-fold molar excess or 20 µl of the suspended monomaleimide-Nanogold was added to the 9.2 µg
of purified receptor and incubated at 4 °C in the dark overnight.
The insulin receptor·monomaleimide-Nanogold complex was dialyzed in a
50-µl dialysis button using a 50,000 molecular weight cut-off
membrane against 200 ml of 30 mM HEPES, 0.6% -octyl
glucoside, 50 mM NaCl for 7.5 h at 4 °C. The
dialysate was replaced with 400 ml of the same buffer and dialyzed
overnight at 4 °C, replaced again with 300 ml of the same buffer,
and dialyzed for 9 h at 4 °C. Maleimide-gold-labeled insulin
receptor was prepared for cryo-electron microscopy or receptor reconstitution.
22 insulin receptor was added to 27.5 µl of large unilamellar eggPC vesicles (2 mg/ml) in a total volume of
55.0 µl and dialyzed in 50-µl dialysis buttons against 10 ml of 30 mM HEPES, 100 mM NaCl, 0.6% -octyl
glucoside, and 0.01% NaN3 overnight at 4 °C. The sample
was then sequentially dialyzed at 4 °C against 10 ml of 30 mM HEPES, 100 mM NaCl, 0.3% -octyl glucoside, and 0.01% NaN3 for 3 h; 10 ml of 30 mM HEPES, 50 mM NaCl, 0.15% -octyl
glucoside, and 0.01% NaN3 for 3 h; 20 ml of 30 mM HEPES, 25 mM NaCl, and 0.01%
NaN3 without -octyl glucoside for 3 h. The dialysis
continued at 4 °C against 100 ml of 30 mM HEPES, 10 mM NaCl, and 0.01% NaN3 overnight, followed by
250 ml of the fresh buffer for 7 h, and finally against 500 ml of
30 mM HEPES, 10 mM NaCl, and 0.01%
NaN3 overnight. The reconstituted sample was prepared for
electron microscopy.
-octyl glucoside, and 0.01%
NaN3. Carbon-coated copper grids (400-mesh Gilder grids)
(Ted Pella Inc., Redding, CA) were glow-discharged in air three times
at 2.5 mA for 29 s using a Balzers Union glow discharge apparatus
immediately prior to use. 4 µl of diluted sample were incubated on a
grid for 2 min and rinsed with 8-12 drops of water. 4 drops of 1%
uranyl acetate (Electron Microscopy Sciences, Fort Washington, PA) were
then applied to the grid, and the final drop was allowed to incubate for 30 s before blotting off the excess stain.
174 °C. Samples were viewed using a Philips CM12 transmission electron microscope under low dose conditions, and electron micrographs were recorded on Kodak Electron Image Film SO-163 (magnification, ×75,000). The high tension setting was 100 kV, using a 50-µm
condenser aperture and a 30-µm objective aperture. The camera was set
at 0.701 s, and the beam intensity was adjusted to a meter reading of
1.47 s. Micrographs were recorded at 1.7 µm underfocus for protein or vesicles. Micrographs of gold-labeled protein or vesicles were recorded at both 1.7 µm and 0.5 µm. 1.7 µm micrographs were taken first and were optimal for visualizing protein and/or vesicles. 0.5 µm micrographs were taken second and were optimal for
visualizing gold. Negatives were developed as described above. The
total electron dose was less than 40 e/Å2.
1.0. The criteria for particle
selection required that the images have good contrast, with minimal
astigmatism, an even ice layer across the selected region, proper
underfocus, and three identifiable "arms" per receptor. The
contrast corrected images were visualized in WEB, and individual particles were selected. The selected images were normalized from 0 to
1, masked, and organized in a montage.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
half-receptor by Western blotting (Fig.
1B, lanes 3-5). The half-receptor could be separated from the heterotetrameric receptor by ion-exchange chromatography (data not shown) and is not present in the purified product (Fig. 1, A, lane 6, and
B, lane 6). Functionality of the fully
purified insulin receptor was confirmed by autophosphorylation assays
(Fig. 1C). Insulin receptors were incubated in the absence and presence of insulin with ATP and the appropriate metal ions. The
phosphotyrosine content of the insulin receptor was detected by
blotting with an anti-phosphotyrosine antibody. Fig. 1C
illustrates that the insulin-dependent phosphorylation of the
fully purified receptor corresponds to that of crude wheat
germ-purified receptor controls (Fig. 1C). These data
confirm that the human insulin receptor obtained from this extensive
purification protocol is intact as measured by SDS-PAGE, Western
blotting, and functionality, the latter determined by
autophosphorylation assays.
View larger version (70K):
[in a new window]
Fig. 1.
The insulin receptor purification protocol
results in pure, functionally active heterotetrameric insulin
receptor. A, each step of the purification results in
less contaminating protein and a more prominent
22 heterotetrameric insulin receptor
band. Lanes were loaded for approximate equal amounts of
heterotetrameric insulin receptor. B, representative Western
blot from the insulin receptor purification (antibody = anti-insulin receptor -subunit). The final purification product
corresponding to 350 kDa is 22
heterotetrameric insulin receptor. The band corresponding to 200 kDa in
lanes 3-6 is half-receptor. C,
autophosphorylation assay. In the presence of insulin, phosphotyrosine
content of the fully purified receptor is comparable to that of control
wheat germ-purified insulin receptor. Lane 1,
membranes; lane 2, Triton-solubilized proteins;
lane 3, Sephacryl 400 pools; lane
4, wheat germ agglutinin-agarose eluant; lane
5, MonoQ pools; lane 6, Superose 6 pools.
Detergent-solubilized Insulin Receptor
The purified, functionally active, heterotetrameric insulin
receptor was visualized in the detergent-solubilized form using negative stain electron microscopy under low electron dose conditions. Several stains were used including sodium phosphotungstate and methylamine tungstate (data not shown); however, uranyl acetate provided the highest contrast and the most reproducible results. There
were no large protein aggregates evident in the stained samples;
however, occasionally, small clusters containing two or three receptors
were seen. Regions with light, even stain were optimal for
visualization of individual receptor particles with minimal detergent
artifact. Selected receptors from the resulting micrographs are shown
in Fig. 2. The negatively stained insulin receptors take on the same Y- or T-shaped appearance reported previously (19). As illustrated in Fig. 2A, there is
considerable variation among individual receptors. Some appear as a
perfect Y (Fig. 2A, 1), while others appear as a
perfect T (Fig. 2A, 15); however, most of the
receptors do not conform precisely to either shape and demonstrate
"characteristics" of these two shapes (Fig. 2A,
7, 11, 12, 17,
18, 20, and 22).
|
Purified receptors were incubated in the presence of 100-fold molar excess of insulin prior to staining. The presence of insulin did not appear to affect the general staining characteristics of the insulin receptor. Unlike the results reported by Johnson et al. (30), insulin did not appear to induce aggregation of the purified holoreceptors. Fig. 2B displays selected individual insulin receptors in the presence of insulin. These receptors show the same Y- and T-like characteristics observed in the absence of insulin (Fig. 2A). There are receptors that appear like perfect Y (Fig. 2B, 4) and T (Fig. 2B, 12) shapes, although the majority of the receptors appear to be variations on these themes. These data demonstrate that insulin receptors exhibit the same general multiformity with and without insulin. In the presence of insulin, the receptors appear to be slightly more globular and less variable in structure. Quantitative measurements, however, show that insulin does not affect the overall length of individual receptor arms, nor does it affect the distribution of arm lengths (data not shown). Insulin receptors in the presence of insulin appear to be slightly larger in width than insulin receptors without insulin (data not shown); however, the limited resolution of these images prohibits the conclusion that this difference represents an insulin-induced conformational change.
In order to visualize the insulin receptor in a fully hydrated state,
free of heavy metals and staining artifacts, we used the technique of
cryo-electron microscopy. When preserved in vitreous ice and visualized
using cryo-electron microscopy, the insulin receptor retains the Y- or
T-shaped appearance identified above in negative stain. Fig.
3A is a representative field
of detergent-solubilized receptors visualized using cryo-electron
microscopy. Individual receptors from this field, and others, have been
selected and displayed in Fig. 3B. In the selected
receptors, the Y-like characteristics of the insulin receptor (Fig.
3B, 4, 10, 12,
23, 25, etc.) seem to dominate over the T-like
characteristics (Fig. 3B, 20 and 32), although both are evident. For many of the receptors, the Y-like or
T-like appearance depends greatly on the perception of the individual
observer. Of the 48 representative receptors selected and displayed in
Fig. 3B, no 2 are identical, differing in arm length,
separation angles, or both. Because of the individual receptor
variability, and lack of orienting features, overall receptor
dimensions were not determined. The cryo-electron microscopy data do,
however, confirm the gross insulin receptor structure identified in
negative stain. As illustrated by Fig. 3B, the four individual subunits of the insulin receptor come together to form a
three-armed macromolecular complex.
|
Gold Labeling
Streptavidin-Nanogold-- To address the issue of where insulin binds to the three-armed pinwheel complex identified using cryo-electron microscopy, we have employed an insulin analogue, BBpa insulin (26) and streptavidin-Nanogold. BBpa insulin is a photoactivatable insulin analogue that is covalently cross-linked to the insulin receptor when exposed to UV light (26). Residue B29 of this analogue has a biotin moiety that is accessible to streptavidin binding (26). Using a streptavidin-Nanogold conjugate, we have been able to label the insulin binding site with a 1.4-nm gold particle and visualize the insulin receptor·BBpa-insulin·streptavidin-Nanogold complex by cryo-electron microscopy.
Insulin receptors in the presence of the
BBpa-insulin· streptavidin-Nanogold complex exhibit the same
characteristics that were evident in the detergent-solubilized insulin
receptor alone (Fig. 4). The receptors
are identifiable, three-armed complexes (Fig. 4A,
1, 4, 7, 10, 14,
and 15). Only one gold particle is seen per receptor,
suggesting that only one insulin analogue binds per receptor,
confirming the studies reported by Lee et al. (9). The
Nanogold particles were localized to the tip of one arm, and the
identity of the gold was confirmed by defocus pairs (data not shown).
Because insulin binds to the -subunit of the insulin receptor, the
presence of a gold particle at the tip of one of the arms identifies
that arm as containing the -subunit. Identifying one of the
-subunits does not assist in orienting the receptor subunit
composition with respect to the two-dimensional projection, as this
does not distinguish the identity of the other two arms. The location
of the gold particle does, however, confirm that insulin binds to that
arm and suggests that the insulin binding site is not itself at the arm
tip. Because streptavidin was used as a linker between the gold
particle and the insulin analogue, the true insulin binding site is at
least 5 nm away from the gold particle. Additionally, the lack of
distinct density attributable to the streptavidin molecule in the
gold-labeled receptors suggests that the streptavidin density is
concealed by the receptor density. Taking into account the streptavidin
spacer and the effect of projecting the receptor complex in two
dimensions, it is possible that the insulin binding site lies toward
the center of the receptor molecule in a manner similar to that of the
interaction between growth hormone and the growth hormone
receptor (31).
|
Biotin-Maleimide Labeling--
Previous studies on the
extracellular domain of the insulin receptor report a V-like or short
T-like structure (12). This suggests that two of the arms of our
three-armed structure contain the -subunits, and the third arm
contains both -subunits. Identifying the -subunit enables us to
orient the receptor because the identity of all three arms would be
determined. The -subunit of the insulin receptor can be
distinguished from the -subunit based on its free sulfhydryl content
(32-34). We incubated purified insulin receptor with a
biotin-maleimide conjugate and detected this conjugate with
streptavidin-linked horseradish peroxidase. Fig.
5 shows that in the absence of reducing
agent, the heterotetrameric insulin receptor is labeled by the
maleimide conjugate. Complete reduction of the holoreceptor into its
component subunits demonstrates that greater than 95% of the labeling
of the 22 receptor is located in the
-subunit with a trace corresponding to incorporation into the
-subunit (Fig. 5A, lane 4).
Compared with the extremely strong -subunit band, this -labeling
is inconsequential and is most likely a result of some small degree of
disulfide reduction. The identity of the individual subunits was
confirmed with anti-insulin receptor -subunit antibody and
anti-insulin receptor -subunit antibody (Fig. 5B,
lane 4). These data confirm the previous reports that only the -subunit is accessible to N-ethylmaleimide
(NEM) labeling in the absence of reducing agents (32-34).
|
Maleimide-Gold Labeling--
Using monomaleimide-Nanogold, we
specifically labeled the -subunit of the insulin receptor for
visualization using cryo-electron microscopy. Unlike the
streptavidin-Nanogold used to label the -subunit of the insulin
receptor, monomaleimide-Nanogold does not have a large protein spacer.
When the maleimide moiety is covalently attached to the sulfhydryl of a
protein, the center of the gold particle is only 2 nm from the cysteine
side chain (35). This property enables more direct labeling of the
insulin receptor than the insulin·biotin·streptavidin complex used
to label the -subunit.
Fig. 6 shows representative
maleimide-Nanogold-labeled insulin receptor heterotetramers. The
receptors were oriented using the SPIDER image processing package (29)
to position the gold particle identifying the -subunit. In these
images, the insulin receptors are oriented such that the gold is at the
6 o'clock position. As is evident from these images, even with this
orienting label, the receptor takes on a variety of conformations,
demonstrating both Y-like and T-like characteristics of the three armed
complex as described above for both the unlabeled and
-subunit-labeled receptors. Only one gold particle was evident per
three-armed receptor complex, and again, the gold particle was
routinely localized at the tip of a single arm. Previous quantitative
analysis of NEM labeling dimers indicates that one NEM binds per
dimer (32); however, analysis of NEM labeling
22 heterotetramers indicates that there
is 1.13 mol of NEM labeling/mol of insulin binding activity, indicating
that one NEM binds per tetramer (34). These studies suggest that either
only one sulfhydryl in the heterotetrameric receptor is accessible to
maleimide labeling, or that there is steric interference from the first
gold particle preventing the binding to a second maleimide-gold
label.
|
Insulin Receptor Reconstitution Visualized Using Negative Stain Electron Microscopy
Detergent-solubilized forms of isolated membrane proteins
frequently lose some of the functional activity present in the membrane bound forms (reviewed in Refs. 36 and 37). This loss of function can be
indicative of minor structural changes that result from the absence of
membrane interactions. Reconstituting the insulin receptor into
pre-formed phospholipid vesicles provided two important experimental
advances. First, it enabled us to study the insulin receptor in a model
membrane environment that more closely approximates the physiologic
state of the receptor. Second, like the maleimide-gold labeling,
reconstituting the receptor enabled us to orient the receptor in a
two-dimensional plane by identifying the -subunit-containing arm.
Sequence analysis has identified the -subunit as the only subunit
that crosses the membrane (38, 39). We have
created a population of large unilamellar
egg phosphatidylcholine (eggPC) vesicles by extrusion (27) (Figs.
7A and 8A) and used
these vesicles for insulin receptor reconstitution. The pre-formed
unilamellar eggPC vesicles were incubated with detergent-solubilized
purified insulin receptor and 20 mM -octyl glucoside
prior to detergent removal over a 48-h period using stepwise dialysis.
The resulting reconstituted receptors were visualized by both negative
stain and cryo-electron microscopy.
|
|
Reconstituted insulin receptors were negatively stained in 1% uranyl acetate and visualized under low electron dose conditions. These samples did not stain as uniformly and reproducibly as the detergent-solubilized receptors; however, there were consistently regions of light, even staining that enabled adequate visualization of the reconstituted receptors (Fig. 7B). Individual reconstituted receptors were selected from representative micrographs and displayed in Fig. 7C. These receptors are more difficult to see in negative stain than are their detergent-solubilized counterparts (cf. Fig. 2). Fig. 7C (1) demonstrates the typical Y-like structure protruding from the vesicle surface, and several other receptors demonstrate the two armed V-like structure that results when one of the Y arms is hidden (Fig. 7C, 15, 19, and 20). Some regions clearly contain more than one reconstituted receptor per vesicle (Fig. 7C, 12). Other regions are highly suggestive of two adjacent receptors per vesicle; however, both receptors are not clearly demarcated (Fig. 7C, 5 and 14). Most of the negatively stained reconstituted receptors are seen as stain-excluding regions extending from the vesicle surface providing minimal structural detail.
Insulin Receptor Reconstitution Visualized Using Cryo-electron Microscopy
Reconstituted insulin receptors preserved in vitreous ice were visualized by cryo-electron microscopy under low electron dose conditions (Fig. 8B). Cryo-electron microscopy overcomes many of the limitations of negative stain, and the reconstituted receptor images provide much more detail (Fig. 8C) than the negatively stained images (Fig. 7C). The original vesicle population was fairly heterogeneous; however, the reconstitution procedure seemingly resulted in an increase in the proportion of multilamellar vesicles (data not shown). Free protein was occasionally evident in the background. The criterion we used for selecting reconstituted receptors required that there be an evident membrane perturbation at the site corresponding to the receptor's transmembrane region. Use of this strict criterion for receptor selection limited the receptors identified as being reconstituted, but avoided misclassification of the free protein. Representative reconstituted receptor images were selected and low pass-filtered for contrast enhancement. The resultant images are displayed in Fig. 8C.
Fig. 8C shows the reconstituted insulin receptors as the same three-armed complex that we have identified for the detergent-solubilized insulin receptors (Fig. 3B). The reconstituted insulin receptors demonstrate one of the arms traversing the vesicle bilayer and the other two arms removed from the vesicle surface (Fig. 8C). Most of the reconstituted receptors exhibit more Y-like characteristics (Fig. 8C, 1, 2, 5, and 11); however, there are still some receptors that exhibit more T-like characteristics (Fig. 8C, 45 and 47). As with the detergent-solubilized receptors, the reconstituted receptors still display extremely variable structures. Anchoring the receptor transmembrane domain in a model membrane does not appear to limit the receptor orientational variability to any great extent. Of the 48 reconstituted receptors in Fig. 8C, no two images are identical. These receptor images do not contain any distinct globular domains in contrast to the report by Tranum Jensen et al. (20), identifying globular domains at the arm tips of negatively stained insulin receptors. The images displayed in Fig. 8C each contain one clear reconstituted receptor; however, there are some views that do contain additional receptors in the fields (Fig. 8C, 5, 11, and 24). These "single" images were selected to illustrate clear receptor structure. More than one receptor, however, can be present on a single vesicle, and the presence of these multiple receptors in close proximity does not appear to limit the variability of the resulting images (data not shown).
Reconstituted -Subunit-labeled Insulin Receptors
We have oriented the detergent-solubilized three-armed receptor
complex with respect to subunit composition by gold labeling the
-subunit as described above. The reconstituted insulin receptors were already orientationally defined based on identification of the
-subunit-containing arm as the only arm able to cross the membrane
(38, 39). Our reconstituted 22
heterotetrameric receptor images, however, did not clearly define the
intravesicular component of the receptor. We applied a -subunit
labeling technique, similar to that used for the detergent-solubilized
insulin receptor, to the reconstituted 22
heterotetrameric receptor to identify the intravesicular component of
the receptor.
The -subunit of purified insulin receptor was labeled with
monomaleimide-Nanogold and reconstituted into pre-formed unilamellar eggPC vesicles. Because the receptor was gold-labeled prior to reconstitution, the gold-labeled -subunit could be either
intravesicular or extravesicular. The reconstituted labeled receptor
was visualized by cryo-electron microscopy, and data were recorded in
defocus pairs (1.7 µm and 0.5 µm) to confirm the presence of
gold attached to the receptor. The criteria used for identification and
selection of the reconstituted gold-labeled receptor required that the
receptor have clear protein at a defocus of 1.7 µm and clear
corresponding gold at a defocus of 0.5 µm. Both components,
however, did not have to be visible at a single defocus value. There
also had to be detectable membrane disturbance at the region of
receptor insertion. Individual receptors meeting these criteria were
selected and displayed in Fig. 9.
|
The gold-labeled reconstituted insulin receptors displayed in Fig. 9
demonstrate the same three-armed complex described for detergent-solubilized and reconstituted insulin receptors. The gold
particle (seen as a black dot in the
primed numbers) can easily be identified at the
0.5 µm defocus value (Fig. 9A). The gold particle is
attached to the one receptor arm that is part of the membrane-spanning
arm, confirming that this is the -subunit-containing arm. The
position of the gold particle with respect to the vesicle membrane is
not constant. For example, the gold particle in Fig. 9A
(1') is 2.4 nm from the vesicle membrane, while the gold
particle in Fig. 9A (3') is flush against the
membrane, and the gold particle in Fig. 9A (4')
appears to be within the lipid bilayer. The precise cysteine residue
that is labeled by maleimide conjugates has yet to be identified. It
has been proposed that the accessible cysteine on the -subunit lies
near the ATP binding site because NEM inhibits insulin receptor kinase
activity (40) and binding of nucleotides to the insulin receptor
decreases NEM labeling (32). By assuming that the gold particle covers
at least the kinase region of the receptor and potentially part of the
C terminus, we have been able to approximate the length of the
intravesicular component. The positioning of the gold particle on the
reconstituted images must be considered in terms of the resultant
two-dimensional projection of a three-dimensional object. Just as the
true receptor arm length is represented by the longest projection, the
true distance between membrane and gold particle is represented by the
longest projection. The longest projection, represented by Fig.
9A (7'), is 4.3 nm when measured from the vesicle
membrane surface through the entire gold particle.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Insulin binding to the insulin receptor initiates a cascade of
cellular events that are responsible for regulating cell metabolism, proliferation, and growth (reviewed in Refs. 2 and 41). We have been
using the tools of electron microscopy to investigate the structure of
the insulin receptor. Negative stain and cryo-electron microscopy both
show the detergent-solubilized insulin receptor as a three-armed
pinwheel structure. The different orientations and subtle variations
are consistently represented by both techniques. Cryo-electron
microscopy, however, provides more structural detail and a more
accurate representation of the receptor in an aqueous environment. The
gross structural appearance of the detergent-solubilized 22 heterotetrameric receptor correlates
with the previously reported Y-like or T-like appearance of the insulin
receptor extracellular domain (12) and holoreceptor (19), respectively.
The individual images in Figs. 2 and 3C exemplify the
considerable variability that the insulin receptor demonstrates when
visualized by electron microscopy.
Electron microscopy involves reducing a three-dimensional object (the insulin receptor) to a two-dimensional projection (the resulting micrograph). The variability seen among receptor images can be explained in one of two ways. The first explanation is that there is inherent flexibility within the protein itself, allowing it to freely adopt multiple conformations. The second explanation involves the orientation of the receptor in the ice layer. Because the ice layer thickness of a sample viewed by cryo-electron microscopy is on the order of 100 nm (much larger than the dimensions of the receptor), the detergent-solubilized insulin receptor has a full 360° of rotational freedom in that ice layer. This rotational freedom enables the insulin receptor to exist in innumerable different orientations in the ice layer. Without an appropriate orientational reference, the cause of the apparent flexibility cannot be determined. By using gold labels visible under the conditions of cryo-electron microscopy, we were able to identify the subunits composing the three-armed receptor complex in an effort to elicit an orientational reference.
We have compiled the data from our cryo-electron microscopic studies of the insulin receptor to construct a two-dimensional model (Fig. 10A). Several assumptions were made in composing this model. First, the insulin receptor is presented with Y-like characteristics,
