Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering

Abstract

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.

Keywords: CD44; Cell Adhesion; Cell Adhesion Molecule; Ezrin; Neutron Scattering; Phosphatidylinositol.

FIGURE 1.

The cytoplasmic domain of CD44 is a disordered monomer in solution. A, amino acid sequence of CD44ct. The multiple basic residues that bind PIP2 are highlighted in red. B, gel filtration chromatogram of Trx-CD44ct. C, static light scattering measurements of the molecular mass of Trx-CD44ct at different protein concentrations. D, CD spectra of CD44ct in solution (black line) and in PIP2 (red line). The protein concentration used for CD experiments is 0.1 mg/ml, and PIP2 concentration is 125 μm. E, comparing SANS data of ^dTrx-CD44ct (open black circle) and ^dCD44ct (filled blue square). The concentrations of ^dCD44ct and ^dTrx-CD44ct are 1.5 mg/ml, respectively. F, Guinier plot of ^dTrx-CD44ct (open black circle) and ^dCD44ct (filled blue square). G, P(r) function of ^dTrx-CD44ct and ^dCD44ct in H₂O buffer. As a comparison, P(r) of the crystal structure of thioredoxin (black line, PDB code 2TRX) is shown. H, the three-dimensional shape of ^dCD44ct is docked to that of ^dTrx-CD44ct. The three-dimensional shapes of ^dCD44ct and ^dTrx-CD44ct are reconstructed ab initio from SANS data, respectively, using the program DAMMIN (41). The crystal structure of thioredoxin (PDB code 2TRX) is docked in dTrx-CD44ct envelope.

FIGURE 2.

Comparing the conformation of CD44ct in solution and in complex to PIP2. A, protein concentration normalized SANS data I(Q)/c of ^dCD44 in buffer solution (black filled square) and in PIP2 solution (black open circle). The SANS experiments were performed in 20% D₂O at the contrast-matching point of PIP2. The concentration of ^dCD44ct is 1.5 mg/ml, and the concentration of PIP2 is 1.2 mm. B, P(r) function of the SANS data shown in A. C, the normalized Kratky plots of ^dCD44ct in solution. D, the normalized Kratky plots of ^dCD44ct in PIP2 solution. The comparison suggests that, upon binding to PIP2, the conformation of CD44ct changes from a collapsed and disordered globule to aggregates composed of random coil-like chains.

FIGURE 3.

PIP2 is required for CD44ct to bind to full-length Ezrin or to the FERM domain of Ezrin. A, gel filtration analysis of full-length Ezrin(T567D) alone (black), Ezrin(T567D)+CD44ct (red), 1Ezrin(T567D)·10 PIP2 (green), and 1Ezrin·1CD44ct·10PIP2 (blue). B, gel filtration of FERM domain (black) and FERM+CD44ct (red). C, gel filtration of FERM alone (black), 1FERM·10PIP2 (blue), and 1FERM·1CD44ct·10PIP2 (red). D, gel filtration of CD44ct alone (black), CD44ct·100DHPC (red), and CD44ct·10PIP2 (blue). For CD44ct, the absorption of UV at 280 nm is low because the theoretical extinction coefficient is zero. E, pulldown experiments of CD44ct with FERM in the absence and presence of PIP2 using the His₆-CD44ct as bait. F, ITC experiments of CD44ct binding to FERM in solution.

FIGURE 4.

Contrast variation SANS from the PIP2·^dCD44ct·^dFERM complex. A, SANS I(Q) of the ^dCD44ct·^dFERM complex in 326 μm PIP2 and in 0, 20, 40, 60, and 100% (v/v) D₂O buffer, at protein concentrations of 45.5, 24.7, 34.2, 34.2, 22.4, and 70.8 μm, respectively. Note the ^dFERM used in this set of experiment contains an N-terminal fusion tag of 27 residues (see Table 1 for theoretical molecular mass). B, P(r) function of the SANS data shown in A. C, normalized I(0)^0.5 versus the neutron scattering length density of the buffer ρ_o at different D₂O concentrations (see Equation 1). D, Stuhrmann plot of Rg² versus 1/Δ̄ρ, where Δ̄ρ is the average neutron scattering length density contrast of the whole complex against the buffer.

FIGURE 5.

Contrast variation SANS from the PIP2·^dCD44ct·^hFERM complex. A, SANS I(Q) of ^dCD44ct·^hFERM complex in 600 μm PIP2 and in 0, 10, 20, 40, 80, and 100% D₂O buffer, at protein concentrations of 52, 48.1, 51.8, 52.1, 54.2, and 58.8 μm, respectively. The lines are fits to the experimental data when using the multiphase program MONSA (48) to reconstruct the three-dimensional shape of the complex. Note the ^dFERM used in this set of experiment does not contain the fusion tag (see Table 1 for theoretical molecular mass). B, P(r) functions of the SANS data shown in A. C, normalized I(0)^0.5 as a function of ρ_o. D, Kratky plots of PIP2·^dCD44ct·^hFERM at three contrasts, in 0% D₂O, 40% D₂O (contrast-matching point of ^hFERM), and 100% D₂O (contrast-matching point of ^dCD44ct). E, the image of the PIP2·^hFERM components in the PIP2·^dCD44ct·^hFERM complex at 100% D₂O, which is the contrast-matching point of ^dCD44ct. The image reveals a dimer of FERM domains in the complex.

FIGURE 6.

A and B, static and dynamic light scattering of PIP2·CD44ct complex (A) and PIP2·CD44ct·FERM complex (B). The table shows the molecular mass (Mw) from static light scattering and the hydrodynamic radius R_h and R_h polydispersity (%) from dynamic light scattering.

FIGURE 7.

A, sedimentation velocity analysis of PIP2·CD44ct·FERM complex showing a plot of c(s) distribution versus sedimentation coefficient. The actual data points representing the sedimentation boundary of this complex and the corresponding fit (upper panel of inset) including the residuals for this fit (lower panel of inset) are shown in the inset. B, sedimentation equilibrium analysis of PIP2·CD44ct·FERM complex showing data points and global fits from the same cell in equilibrium at a range of two rotor speeds (10,300 rpm, purple; 18,000 rpm, blue) in the upper panel and residuals for the corresponding fits in the lower panel.

FIGURE 8.

Comparing three-dimensional shapes of the PIP2·^dCD44ct·^dFERM and PIP2·^dCD44ct·^hFERM complexes. A, PIP2·^dCD44ct·^dFERM. The three-dimensional image was reconstructed from the SANS data in 20% D₂O at the contrast-matching point of PIP2 using the program DAMMIN (41). The presented image was obtained by averaging 17 PDB files generated, using the program DAMAVER (41). The normalized spatial discrepancy values, which measure the reproducibility of the models used in averaging, range from 0.760 to 1.080. B, PIP2·^dCD44ct·^hFERM. The three-dimensional image was reconstructed using the program MONSA (41), assuming PIP2 (red), ^dCD44ct (gold), and ^hFERM (green) of different neutron scattering length densities as three different phases.

FIGURE 9.

Dimerization of full-length Ezrin in PIP2 in the presence of CD44ct. A, SANS of 1.57 mg/ml ^dEzrin(T567D) in 20% D₂O, 1.41 mg/ml ^dEzrin(T567D) in 2.3 mm PIP2 in 20% D₂O, and 0.8 mg/ml ^hCD44ct/^dEzrin(T567D) in 2.3 mm PIP2 in 20% D₂O, at the contrast-matching point of PIP2. B, P(r) of the SANS data shown in A. C, open conformation of a ^dEzrin(T567D) dimer in complex to ^dCD44 in PIP2 reconstructed from SANS data.

FIGURE 10.

A schematic depicting the clustering of CD44ct upon binding to PIP2 and the assembly of a CD44·Ezrin heterotetramer complex in PIP2.