(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 …
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 …
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.
(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 …
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 …
(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 …
(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) …
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 …
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 …
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 …
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 …
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 …
(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 …
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). …
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 …
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 …
(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 …
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 …
(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) …
Coomassie-blue stained reducing SDS-PAGE analysis of purified 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 …
Coommassie-blue stained native gel of phosphorylated and unphosphorylated forms of IGF1R-kin, IGF1R-jmk, and IGF1R-icd.
(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 …
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…
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).
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.
(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, …
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 …
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 |
The absolute value of Km could not be determined but is greater than value stated.
kcat value could not be measured.
Enzymatic values (±s.d.) calculated from duplicate experiments are shown.
See also supplemental figures.
Representative curves of steady-state kinetic analyses for each IGF1R protein characterized.
Each data point was performed in duplicate and is shown separately.
Enzyme behavior is linear with respect to time.
Product/Enzyme plotted vs time (minutes) for each IGF1R protein investigated.
Enzyme behavior is linear with respect to enzyme concentration.
Velocity (nM of product/min) plotted vs enzyme concentration (nM) for each IGF1R protein investigated.