How IGF-1 activates its receptor
- Johns Hopkins University School of Medicine, United States
- University of the Sciences, United States
- DE Shaw Research, United States
- Johns Hopkins University, United States
- Columbia University, United States
Figures
Figure 1 with 3 supplements
The extracellular domain of IGF1R autoinhibits IGF1R activity.
(A, B) Schematic and cartoon representations of IGF1R. Numbering refers to human IGF1R excluding the signal sequence. Disulfide bonds are indicated by black diamonds. One αβ subunit is shown in …
Figure 1—figure supplement 1
Residues on IR important for ligand binding.
Worm diagram of the unliganded IR ECD with one αβ subunit colored white and the other orange. Residues implicated in forming the Site 1 binding surface are shown as cyan spheres and residues …
Figure 1—figure supplement 2
Phosphorylation of the ICD.
Cartoon of IGF1R-icd in the inactive or active, phosphorylated conformations. The activation loop is indicated by a purple line and phosphorylation sites as pink circles.
Figure 1—figure supplement 3
Activity assay of IGF1R TM-icd.
(A) Cartoon of IGF1R Fn3-TM-icd used in the cell-based assay (left). Western blots of normalized cell lysates of HEK293 cells expressing IGF1R-fl (FL), wild-type IGF1R Fn3-TM-icd (WT), or an IGF1R …
Figure 2
Conserved IR ECD dimer interaction.
Surface representations of (A) the unliganded IR ECD dimer (PDB 3LOH; Smith et al., 2010) and (B) the IR ECD fragment bound to Insulin (PDB 3W14; Menting et al., 2013). Domains of one αβ subunit are …
Figure 3
Ligand-induced conformational change in the IR ECD.
(A) Surface representation of the unliganded IR ECD (PDB 3LOH; Smith et al., 2010) oriented to show the L1:Fn2'-3' interface. The hinge between CR and L2 is indicated by a cyan circle. Cartoons of …
Figure 4 with 5 supplements
The IGF1R ECD maintains TM separation.
(A) Cartoon of the IGF1R ECD-TM-fp variant utilized in the live cell FRET assay (top). Frequency distribution of FRET efficiency values per membrane pixel of IGF1R ECD-TM-fp in the presence (green) …
Figure 4—figure supplement 1
Apparent FRET efficiency measured in membrane-derived vesicles.
Measured FRET efficiencies between isolated IGF1R TM-fp (pink circles), IGF1R ECD-TM-fp with IGF1 (green circles) or without IGF1 (white circles), IGF1R ECD-ΔL1-TM-fp (blue circles), and IGF1R …
Figure 4—figure supplement 2
Ratio of donor to acceptor molecules in membrane-derived vesicles.
Each data point represents the measured number of donors and acceptors per square micron measured for a single vesicle from experiments on IGF1R TM-fp (pink circles), IGF1R ECD-TM-fp with IGF1 …
Figure 4—figure supplement 3
Intrinsic FRET efficiency in vesicles, corrected for proximity FRET and varying donor-to-acceptor ratios.
Intrinsic FRET efficiencies between isolated IGF1R TM-fp (pink circles), IGF1R ECD-TM-fp with IGF1 (green circles) or without IGF1 (white circles), IGF1R ECD-ΔL1-TM-fp (blue circles), and IGF1R …
Figure 4—figure supplement 4
The TMs associate in IGF1R ECD-ΔL1-TM-fp.
Cartoons of IGF1R ECD-TM-fp and IGF1R ECD-ΔL1-TM-fp used in FRET assays (left). The intrinsic FRET of IGF1R ECD-ΔL1-TM-fp (blue) in vesicles plotted as a function of receptor concentration and fit …
Figure 4—figure supplement 5
FRET efficiency in live cells of isolated TMs.
A Cartoon of the IGF1R TM-fp utilized FRET assays (left). The frequency distribution is plotted of FRET efficiency values per membrane pixel in live cells of IGF1R TM-fp (pink) (right). The data for …
Figure 5 with 3 supplements
IGF1R TMs associate.
(A) Sequence alignment of human IGF1R and IR extracellular juxtamembrane and TM regions. The position of the TM is indicated above the alignment. Bold lettering highlights residues targeted for …
Figure 5—figure supplement 1
Conservation of extracellular juxtamembrane and TM regions in IR family members.
Weblogo plot showing conservation of the extracellular juxtamembrane and TM residues of 175 non-redundant sequences of Insulin family receptors generated in a BLAST search (Crooks et al., 2004). …
Figure 5—figure supplement 2
P911 is not required for proper IGF1R activation.
IGF1R proteins were immunoprecipitated from normalized cell lysates in the presence or absence of IGF1 and analyzed by Western blots probed with either anti-pY or anti-IGF1Rβ. HEK293 cells were …
Figure 5—figure supplement 3
Stable IGF1R TM dimers form during MD simulations.
Reciprocal Cα-Cα distances for selected residues along the IGF1R TM region are shown as a function of time during the simulation. A stable dimer of IGF1R TMs forms after ∼2 μs in which a contact is …
Figure 6 with 1 supplement
TMs associate in active IGF1R-fl.
(A) Cartoon of IGF1R-fl with cysteine substitutions (top). Western blots of immunoprecipitated normalized cell-lysates from untransfected cells (U) or cells transfected with IGF1R-fl (WT) or …
Figure 6—figure supplement 1
Matching migration patterns of wild-type and H905C IGF1R-fl.
HEK293 cell lysates normalized to total protein concentration from cells expressing either IGF1R-fl (WT) or the H905C variant (905C) were run on non-reducing and reducing SDS-PAGE and analyzed by …
Figure 7 with 3 supplements
IGF1 stimulates autophosphorylation.
(A) Bar graphs of kcat (min−1) measurements are shown for phosphorylated forms of indicated IGF1R proteins. Diagrams of the specific fragments and the fold differences in activation are shown. (B) …
Figure 7—figure supplement 1
Purity of IGF1R-fl.
Coomassie-blue stained reducing SDS-PAGE analysis of purified IGF1R-fl.
Figure 7—figure supplement 2
IGF1 does not co-purify with IGF1R-fl.
To investigate if IGF1 from the growth medium co-purified with IGF1R-fl, we compared a fixed amount of the purified receptor sample to known quantities of purified IGF1 by Sypro-Ruby stained …
Figure 7—figure supplement 3
Analysis of phosphorylation states of IGF1R fragments.
Coommassie-blue stained native gel of phosphorylated and unphosphorylated forms of IGF1R-kin, IGF1R-jmk, and IGF1R-icd.
Figure 8 with 3 supplements
Surface mutations on the IGF1R kinase domain disrupt receptor activation.
(A) Anti-pY and anti-IGF1Rβ Western blots of tagged IGF1R proteins immunoprecipitated from cells incubated in the presence or absence of IGF1. HEK293 cells were transiently transfected with IGF1R-fl …
Figure 8—figure supplement 1
Buried surface analysis of crystal lattice pairs of IR and IGF1R kinase domains.
A scatter plot of the buried surface area (Å2) of each crystal lattice pair of IR or IGF1R kinase domains. A dashed line (red) at 800 Å2 represents the minimal threshold of a biological interface (Ba…
Figure 8—figure supplement 2
Surface analysis of IGF1R kinase domain.
The IGF1R kinase displayed in surface representation showing the location of each cluster colored according to the table of alanine substitutions made for each cluster (PDB 1P4O, Munshi et al., 2002).
Figure 8—figure supplement 3
Analysis of the autophosphorylation of IGF1R kinase clusters.
Autophosphorylation of IGF1R-kin wild-type (WT), cluster 1 (C1) and cluster 4 (C4) as a function of time monitored by anti-pY Western-blot. All lanes shown are from the same blot.
Figure 9 with 1 supplement
Model of IGF1R activation.
(A) Cartoon model of IR family activation. The IDs are shown as black lines in the ECDs, the disulfide linkages as black diamonds, phosphorylation as pink circles, activation loops as purple lines, …
Figure 9—figure supplement 1
Linkage of αCTs contributes to negative cooperativity through destabilization of the second L1’:Fn2'-3' site following ligand binding.
Cartoon model depicting the destabilization of the L1':Fn2-3 interaction after ligand binding at the first L1:Fn2'-3' site. The IDs are shown as black dashed lines, the disulfide linkages as black …
Author response image 1
(A) IR family schematic and (B) cartoon representation. Disulfide bonds indicated by black diamonds.
Tables
Table 1
| enzyme pY | Kmapp ATP (μM) | Kmapp Peptide (μM) | kcat (min−1) | kcat/Kmapp(ATP) | |
|---|---|---|---|---|---|
| IGF1R-fl + IGF1 | + | 82 ± 11 | 48 ± 8 | 459 ± 17 | 5.6 |
| IGF1R-fl + IGF1 | − | 356 ± 61 | 68 ± 19 | 44 ± 3 | 0.1 |
| IGF1R-fl | + | 89 ± 18 | 91 ± 8 | 749 ± 22 | 8.5 |
| IGF1R-fl | − | 172 ± 35 | 66 ± 17 | 62 ± 5 | 0.4 |
| IGF1R-icd | + | 79 ± 9 | 110 ± 12 | 385 ± 13 | 4.9 |
| IGF1R-icd | − | >1000* | >1000* | N.D.† | 0.0013 |
| IGF1R-jmk | + | 68 ± 12 | 52 ± 5 | 363 ± 20 | 6.9 |
| IGF1R-jmk | − | >1000* | >1000* | N.D. † | 0.0029 |
| IGF1R-kin | + | 115 ± 24 | 114 ± 26 | 98 ± 6 | 0.85 |
| IGF1R-kin | − | >2000* | >1250* | N.D. † | 0.0019 |
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*
The absolute value of Km could not be determined but is greater than value stated.
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†
kcat value could not be measured.
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‡
Enzymatic values (±s.d.) calculated from duplicate experiments are shown.
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§
See also supplemental figures.
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Table 1—source data 1
Representative curves of steady-state kinetic analyses for each IGF1R protein characterized.
Each data point was performed in duplicate and is shown separately.
- https://doi.org/10.7554/eLife.03772.022
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Table 1—source data 2
Enzyme behavior is linear with respect to time.
Product/Enzyme plotted vs time (minutes) for each IGF1R protein investigated.
- https://doi.org/10.7554/eLife.03772.023
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Table 1—source data 3
Enzyme behavior is linear with respect to enzyme concentration.
Velocity (nM of product/min) plotted vs enzyme concentration (nM) for each IGF1R protein investigated.
- https://doi.org/10.7554/eLife.03772.024
