Implications of the differing roles of the β1 and β3 transmembrane and cytoplasmic domains for integrin function
- Vanderbilt University Medical Center, United States
- Vanderbilt Medical Center, United States
- Max Planck Institute of Biochemistry, Germany
- Veterans Affairs Hospital, United States
Figures
Figure 1
The β1-K752E mutation decreases collecting duct cell adhesion to collagens.
(A) The sequences of the β1 and β3 TM/CTs are annotated. The highly conserved lysine (K752 in integrin β1and K716 in integrin β3) is colored in red. The β3 TM-only construct used in previous studies …
Figure 2 with 2 supplements
The β1 and β3 transmembrane and cytosolic domains have distinct structures.
(A) 800 MHz 1H-15N TROSY spectrum of the WT integrin β1 TM/CT with peak assignments shown. This spectrum was collected at 45°C and the sample contains 500 µM β1 TM/CT (0.57 mol%), 20% q = 0.3 …
Figure 2—figure supplement 1
900 MHz 1H,15N-TROSY spectra of integrin β1 TM/CT.
900 MHz 1H,15N-TROSY spectra of integrin β1 TM/CTat 45o C in four different bicelle mixtures: (A) Cyclofos-6/DMPC, (B) D7PC/DMPC, (C) D6PC/POPC, (D) D6PC/DMPC. The NMR samples contain ~500 µM β1 …
Figure 2—figure supplement 2
NMR chemical shifts to determine alpha helical content of the integrin β1 and β3 TM/CT.
(A) TALOS-N analysis of backbone 13C NMR chemical shifts to determine α-helical content of the integrin β1 and β3 TM/CT. Sample conditions were as described in the captions for Figure 2A and B. (B) …
Figure 3 with 1 supplement
Examination of the bilayer topology of integrin WT β1 TM/CT and two mutants (K752E, K752R) in bicelles using NMR and paramagnetic probes.
Peak intensity changes in the 600MHz 1H-15N TROSY spectra are reported for the integrin WT β1 TM/CT and two mutants (K752E, K752R) as induced either by 4 mol% 16-DSA as the hydrophobic paramagnet or …
Figure 3—figure supplement 1
1H,15N-TROSY spectral overlays of integrin β1 TM/CT in D6PC/DMPC bicelle with no paramagnetic probe (black) and with either 10 mM Gd-DTPA (red) or 4 mol% 16-DSA (red).
Panels A–B are for integrin β1 K752E and panels C–D are for the K752R mutant form. The NMR samples contain ~500 µM β1 TM/CT, 20% q = 0.3 D6PC/DMPC bicelles, 1 mM EDTA 250 mM IMD, at pH 6.5, with 10% …
Figure 4 with 1 supplement
Examination of the bilayer topology of isolated WT integrin β3 TM and of β3 K716E TM in bicelles using NMR and paramagnetic probes.
Paramagnetic probe-induced intensity changes are reported for the peaks in 900 MHz 1H-15N TROSY spectra of WT integrin β3 TM and β3 K716E TM at 45°C: (A) 1 mM Mn-EDDA, (B) 10 mM Gd-DTPA, (C) 10 mM …
Figure 4—figure supplement 1
900 MHz 1H,15N-TROSY spectral overlays of WT integrin β3 TM in D6PC/POPC/POPS (2:1) bicelles with no paramagnetic probe (black) and with either 10 mM Gd-DTPA (red) (A), 1 mM Mn-EDDA (red) (B), both 10 mM Gd-DTPA and 10 mM EDTA (red) (E), or with 4 mol% 16-DSA (red) (F).
Panels C–D and G–H are the corresponding overlays for the integrin β3 K716E TM-only. The samples contained ~300 µM β3 TM, 20% q = 0.3 D6PC/POPC/POPS (2:1) bicelles, 1 mM EDTA 250 mM IMD at pH 7.4, …
Figure 5 with 3 supplements
Examination of the bilayer topology of WT integrins in bicelles using NMR and paramagnetic probes.
Peak intensity changes are reported as induced by 10 mM Gd-DTPA (A) or 4% 16-DSA (B) in the 900MHz 1H-15N TROSY spectra of WT integrin β3 TM/CT and of the K716E mutant at 45°C in 20% q = 0.3 …
Figure 5—figure supplement 1
TROSY spectra of β3 TM/CT with and without paramagentic probes.
900MHz 1H,15N-TROSY spectral overlay of integrin β3 TM/CT in D6PC/DMPC bicelles with no paramagnetic probe (black) versus 10 mM Gd-DTPA plus 10 mM EDTA (red) (A). 900MHz 1H,15N-TROSY spectral …
Figure 5—figure supplement 2
TROSY spectra of β3 TM/CT with and without paramagentic probes.
900MHz 1H,15N-TROSY spectral overlay of integrin β3 TM/CT in D6PC/POPC/POPS (2:1) bicelles with no paramagnetic probe (black) versus conditions with 10 mM Gd-DTPA plus 10 mM EDTA (red) (A). 900MHz 15…
Figure 5—figure supplement 3
TROSY spectra of of β1 TM/CT with and without paramagentic probes.
900MHz 1H,15N-TROSY spectral overlay of integrin β1 TM/CT in D6PC/POPC/POPS (2:1) bicelle with no paramagnetic probe (black) versus conditions with10mM Gd-DTPA (red) (A). 900MHz 1H,15N-TROSY …
Figure 6 with 2 supplements
Fits of at 1:1 binding model to the data from NMR-monitored titrations of wild type and mutant 15N-labeled integrin β TM/CT subunits by unlabeled wild type α TM/CT subunits, with the best fit Kd determined in each case as shown.
The residue assignments for the TROSY peaks used for these analyses are indicated. In some cases (panels E-H) the binding was so weak that it was only possible to determine a lower limit to the Kd. …
Figure 6—figure supplement 1
Superimposed 900 MHz 1H-15N-TROSY spectra from titrations of 15N-labeled integrin β TM/CTs with unlabeled α subunit TM/CTs: α5β1(WT), α5β1(K752E) αIIbβ3(WT), and αIIbβ3(K716E).
The concentrations of WT β3, WT β1 and the K752E β1 mutant were fixed at 0.17 mol% (150 µM) for all the samples, while the concentration for the K716E β3 mutant was fixed at 0.10% (90 µM). For these …
Figure 6—figure supplement 2
Superimposed 600 MHz 1H-15N-TROSY spectra from titrations of 15N-integrin β TM/CTs with unlabeled α subunit TM/CTs: α1β1(WT), α2β1(WT), α1β1(K752E), and α2K752Eβ1(K752E).
The concentrations of the WT and K752E mutant integrin β1 subunits were fixed at 0.23 mol% (200 µM) for all samples. For these titrations, peak shifts were in some cases observed, indicating subunit …
Figure 7
Use of fluorescence anisotropy to determine Kd for complex formation between α and β integrin TM/CTs.
Measurements were carried out in D6PC/POPC/POPS bicelles (2% total amphiphile, q = 0.3) in 25 mM HEPES buffer pH 7.4 at 35°C. α subunits were labeled with IAEDAN and titrated by unlabeled β …
Figure 8
The β1-K752E mutation decreases collecting duct cell adhesion to fibronectin.
(A) The adhesion of CD cells to fibronectin (0.5 μg/ml) for 1 hr was measured. These assays were carried out in the presence or absence of blocking antibodies directed against the αv or β1 subunits. …
Figure 9
NMR-monitored titrations of integrin β1 TM/CT by the talin-F3 domain.
15N-labeled WT and K752E mutant integrin β1 TM/CT samples were titrated with unlabeled talin1-F3. The concentration of all integrins was 100 µM and the samples contained 5% q = 0.3 D6PC/POPC/POPS …
Figure 10
NMR-monitored titrations of integrin β3 TM/CT by talin.
15N-labeled WT/K716E integrin β3 TM/CT was titrated with unlabeled talin1-F3. The concentration of all integrins was 100 µM. The samples contained 5% q = 0.3 D6PC/POPC/POPS (POPC:POPS = 2:1) …
Tables
Table 1
Dissociation constant (Kd) of integrin β1 and β3 subunits (WT and K752E mutant) with α subunits in D6PC/POPC/POPS (POPC:POPS=2:1) q=0.3 bicelles.
Titration | Kd (mol%) | Monitored subunit | Method |
|---|---|---|---|
α5β1 | 0.17±0.1 | 15N-β1 | NMR |
α5β1 | 0.07±0.02 | α5 | Anisotropy |
α5β1KE | 0.63±0.1 | 15N-β1-KE | NMR |
α5β1KE | 0.80±0.27 | α5 | Anisotropy |
αIIbβ3 | 0.15±0.1 | 15N-β3 | NMR |
αIIbβ3 | 0.09±0.03 | αIIb | Anisotropy |
αIIbβ3KE | 0.51±0.1 | 15N-β3-KE | NMR |
αIIbβ3KE | 0.33±0.05 | αIIb | Anisotropy |
α1β1 | >2.48 | 15N-β1 | NMR |
α1β1 | >3.2 | α1 | Anisotropy |
α1β1KE | >2.48 | 15N-β1-KE | NMR |
α1β1KE | >3.2 | α1 | Anisotropy |
α2β1 | >2.48 | 15N-β1 | NMR |
α2β1 | >3.2 | α2 | Anisotropy |
α2β1KE | >2.48 | 15N-β1-KE | NMR |
α2β1KE | >3.2 | α2 | Anisotropy |
