Cytoskeletal adaptors

Our recent work has shed light on the earliest signals transmitted by chemokines through their G-protein coupled receptors (GPCRs) to distinct leukocyte integrins (scheme 1). In lymphocytes, the integrin adaptor talin1 was found to control major conformational changes of both LFA-1 and VLA-4 integrins implicated in lymphocyte arrest on endothelial ligands. We also found that VLA-4 anchorage to the cytoskeleton is enhanced by the cytoskeletal adaptor paxillin, which cooperates with talin1 to promote the activation of this integrin under external strain but is not essential for chemokine stimulated integrin activation. Experiments with T cells in which talin or paxillin transcription has been silenced suggest that these two adaptors regulate distinct and complementary roles in VLA-4 adhesiveness to the major endothelial ligand VCAM-1.

In addition, we found a major role for the coactivating adaptor, Kindlin-3 (absent in T cells, neutrophils, and platelets from LAD-III patients) in chemokine mediated integrin activation in human leukocytes. In collaboration with R. Fassler (Max Planck Institute for Biochemistry, Martinsried, Germany) we continue to study the role of this adaptor in both human and murine T cells. Interestingly, Kindlin-3 is not required for T cell motility over chemokines, nor for the collapse of microvilli by chemokine signals (Fig. 1). A subgroup of LAD-III patients display defective expression of Kindlin-3 as well as of the PLC-regulated GEF (guanine exchange factor) for Rap-1, CalDAG-GEFI (CDGI), but the loss of Kindlin-3 masks the effect of this deficiency.

Scheme 1 A postulated scheme for rapid chemokine signaling to lymphocyte LFA-1 under shear flow. (Top) A rolling leukocyte tethered to an integrin ligand must encounter juxtaposed chemokine at the site of integrin activation, possibly a single microvillus. A quaternary complex between integrin, ligand, chemokine and G-protein coupled receptor (GPCR) must form within milliseconds to locally activate the integrin ligand complex via a bi-directional signaling event (bottom). Only a fully activated integrin can arrest the rolling leukocyte on the vessel wall while partially activated integrins can participate in rolling adhesions (see Figure 1). Upon initial encounter, endothelial-bound chemokine transduces leukocyte GPCRs signals which convert the inactive (folded) integrin to its extended conformation (step 1). GTP bound RhoA and Rac1 are involved in this step in the case of lymphocyte LFA-1. This critical chemokine-driven inside-out event primes the integrin to transiently bind endothelial ligands on the counter endothelial surface. The various I-domains undergo further conformational shifts upon extracellular ligand binding (step 2), resulting in further integrin activation (outside-in). This ligand-driven step is predicted to result in further separation of the integrin subunit tails, conditional on the presence of talin nearby the ligand occupied integrin, and force transduction (step 3). Clustering of ligand-occupied integrins (not shown) can rapidly follow. Force transduction may lead to talin activation, recruitment of vinculin and crosslinking of integrin-talin complexes to the cortical actin cytoskeleton, all within seconds after initial leukocyte arrest. The additional involvement of Rap1 and its effectors RIAM and RAPL (not shown) in GPCR transduced inside-out activation is suggested for LFA-1 activation by chemokines, whereas PKC is necessary for full VLA-4 activation.

Figure 1 Kindlin-3 is not required for microvillar collapse. CCL21, but not TCR signals, induces similar microvillar collapse in both control and Kindlin-3 null T lymphocytes. Scanning electron microscopy (SEM) images of control and Kindlin-3 null T lymphocytes, intact or stimulated for 2 min with the TCR ligating mAb OKT3 (10 µg/ml) or with soluble CCL21 (10 nM). Bar, 3 αm. Micrographs are representative of >55 cells analyzed in each experimental group.