Aspects of VLA-4 and LFA-1 regulation that may contribute to rolling and firm adhesion

Abstract

Very Late Antigen-4 (CD49d/CD29, alpha4 beta1) and Lymphocyte Function-associated Antigen-1 (CD11a/CD18, alphaL beta2) integrins are representatives of a large family of adhesion receptors widely expressed on immune cells. They participate in cell recruitment to sites of inflammation, as well as multiple immune cell interactions. A unique feature of integrins is that integrin-dependent cell adhesion can be rapidly and reversibly modulated in response to cell signaling, because of a series of conformational changes within the molecule, which include changes in the affinity of the ligand binding pocket, molecular extension (unbending) and others. Here, we provide a concise comparative analysis of the conformational regulation of the two integrins with specific attention to the physiological differences between these molecules. We focus on recent data obtained using a novel technology, based on small fluorescent ligand-mimicking probes for the detection of integrin conformation in real-time on live cells at natural receptor abundance.

Keywords: LFA-1; VLA-4; affinity; cell adhesion; conformation; integrins; rolling; tethering.

Figure 1

Model of VLA-4 integrin conformation and affinity. The bent low affinity state is observed on resting cells (I). Activation by phorbol ester creates a high affinity state lacking molecular extension as detected by a FRET-based approach (II). This conformation results in the slow accumulation of cell aggregates in suspension (Chigaev et al., 2007), with a low tether capture frequency but a long tether duration in the rolling assay (Table 2). The addition of VLA-4 specific ligand to resting cells leads to exposure of a hybrid domain (LIBS) epitope (III). However, the ligand binding affinity remains low [see Figures 2, 4 in Chigaev et al. (2009)]. This state is bent (or at least not fully extended) because a further molecular extension can be detected with a FRET-based approach (Chigaev et al., 2003a, 2004, 2007). Activation through a wild type Gα i-coupled GPCR induces a high affinity extended conformation (IV). This conformation results in the rapid accumulation of cell aggregates in suspension (Chigaev et al., 2007), with high tether capture frequency and long tether duration (Table 2). The low affinity extended (or at least partially extended) conformation (V) can be detected for several minutes after signaling from wild type Gα i-coupled GPCR, because of relatively faster desensitization of the ligand binding affinity than relaxation of the conformation (Chigaev et al., 2007). Conformation V may also result from consecutive stimulation through Gα i-coupled and Gα s-coupled receptors (Chigaev et al., 2008). In suspension this translates into rapid cell aggregation that reaches a steady-state intermediate between resting (I) and Gα i-coupled GPCR activated states (IV) [see Figure 7A in Chigaev et al. (2008)]. A low tether capture frequency and short tether duration was detected in the rolling assay (Table 2) (see text for details). The ligand occupied and extended high affinity state (VI) was detected after Gα i-coupled GPCR activation in the presence of ligand. The molecule affinity and extension were preserved by the use of a non-desensitizing GPCR mutant (Prossnitz, ; Chigaev et al., 2003a, 2007). This conformation results in the rapid formation of a large number of aggregates in cell suspension (Chigaev et al., 2008), with high tether capture frequency and long tether duration in the rolling assay (Table 2). The exposure of the hybrid domain (LIBS) epitope can be also used to determine VLA-4 ligand binding affinity for unlabeled ligands (Chigaev et al., ; Njus et al., 2009).