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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.019117

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Integrins in immunity

Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (95K): [in this window] [in a new window]   Fig. 1. The three conformations of a β2 (ITGB2) integrin. Integrins are heterodimeric glycoproteins comprising non-covalently linked - and β-subunits. Each subunit consists of a large extracellular region, a single hydrophobic transmembrane domain and a short cytoplasmic tail. The extracellular region of the -subunit comprises an N-terminal seven-bladed β-propeller domain followed by three β-sandwich domains (termed thigh, calf 1 and calf 2). Nine of the 18 -subunits (including the four β2-family integrins) also contain an I domain, which is inserted in the upper face of the β-propeller. The β-subunit has an N-terminal cysteine-rich PSI (plexin-semaphorin-integrin) domain, a β-sandwich hybrid domain, a β I-like domain, four integrin EGF-like repeats (I-EGF1 to I-EGF1-4) and a β-tail domain (βTD). In the tertiary structure, the I domain is inserted in the hybrid domain. When present, the I domain is the exclusive site of ligand binding. (A) Bent–inactive. Integrins are bent between I-EGF1 and I-EGF2 in the β-subunit and at a small Ca2+-binding loop, known as the `genu', between the thigh and calf1 domains in the -subunit. Thus, the inactive integrin is in a V shape with the ligand-binding I domain close to the membrane. There is close association between the - and β-subunits in the membrane-proximal region. (B) Extended–intermediate affinity. Inside-out signalling extends the integrin in a `switchblade-like' motion, orientating the I domain away from the membrane for optimal ligand binding. This epitope for the monoclonal antibody (mAb) KIM127, which is located on I-EGF2 and obscured in the bent formation, becomes exposed. The KIM127 epitope thus serves as a marker for the extended β2 integrin. (C) Extended with open conformation–high affinity. Local conformational changes within the and β I domains, potentially generated by shear force, result in the hybrid domain swinging out and the subunit separating at the genu. This remodelling of the I domain ligand-binding site forms the epitope for mAb 24 and causes increased affinity for ligand.

View larger version (27K): [in this window] [in a new window]   Fig. 2. A model of inside-out signalling from agonist to integrin. For integrins to become activated, they need to be triggered by the inside-out signalling cascade. The key target of this pathway is the GTPase Rap1. Agonist signalling leads to an increase in Ca2+ and DAG that activates the Rap1 GEF CalDAG-GEF1, which is a major Rap1 activator in haematopoietic cells. The relocalization of Rap1 to the membrane is regulated by the adaptor protein RIAM. A next step is the recruitment of talin to the β-subunit of the integrin, bringing about the conversion of integrin conformation to one of higher affinity. Kindlin-3 also binds to the β-subunit and aids talin binding.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Outside-in signalling associated with integrins on leukocytes. The inactive β2 and β3 integrins on myeloid cells and platelets, respectively, are constitutively associated with inactive Src kinase. Src is maintained in a C-terminal phosphorylated conformation by Csk kinase. The ligation of integrin to ligand prompts the dephosphorylation of the inhibitory Tyr by phosphatases, such as PTP1B and CD45, dissociation of Csk and autophosphorylation onto the activation loop of Src. Active Src can then dually phosphorylate an ITAM-containing adaptor that has been postulated to be associated with integrin through a linker. The phosphorylated ITAM recruits Syk through its tandem SH2 domains (pale blue). Syk then associates with the integrin β-tail and is in sufficient proximity to Src to be phosphorylated. Active Syk then phosphorylates downstream effectors, such as Vav1, Vav3 and SLP-76.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The location of the LFA-1 conformation zones on a T cell migrating on ICAM1. The leading edge of the T cell expresses low levels of LFA-1 with intermediate affinity for ICAM1 that is recognised by monoclonal antibody (mAb) KIM127 (Stanley et al., 2008). The focal zone expresses higher levels of LFA-1, which is in the high-affinity conformation recognised by both mAb KIM127 and mAb 24 (Smith et al., 2005; Stanley et al., 2008). The uropod at the rear expresses the highest level of LFA-1, but little is known of its binding activity.

View larger version (45K): [in this window] [in a new window]   Fig. 5. The role of LFA-1–-actinin-1 in LN entry and schematic overview of a LN. (A) Effect of β2-actinin-blocking peptide on the migration of mouse T cells into peripheral and mesenteric LNs. The figure shows the effect on lymphocyte migration into LNs of T cells treated with a blocking peptide that consists of the -actinin-1 binding site on the β2 cytoplasmic tail (top panel) linked to membrane-penetrating peptide penetratin-1 (β2-actinin peptide) (Stanley et al., 2008). Mouse LN T cells that had been labelled with the fluorescent dyes CFSE or SNARF-1 were incubated for 30 minutes with either the β2-actinin-blocking peptide or, alternatively, control peptide or Hanks buffered salt solution (HBSS), and injected intravenously into the host for 30 minutes. The numbers of fluorescently labelled T cells that successfully transmigrated into either peripheral or mesenteric LNs were quantified. The β2-actinin-blocking peptide, but not control treatments, severely retarded LN entry, implying a major role for the intermediate-affinity integrin bound to -actinin-1 in this crucial step. (B). Schematic overview of a LN. Leukocytes enter the LN either through the afferent lymph into the subcapsular sinus, or the blood flow through HEVs. T cells migrate in the T-cell zone, thereby contacting resident DCs, which may activate the T cells if they are expressing appropriate antigen. B cells migrate to their follicles seeking an antigen stimulus. As a subsequent stage in their stimulation they encounter T cells at the T-cell–B-cell boundary zone. After migrating through the LN, lymphocytes exit through the medullary sinus into the efferent lymph. The lower panel shows a scheme to highlight integrin dependency (or the lack thereof) during the journey of the lymphocyte through the LN. Green, HEV (entry); purple, T-cell area (intranodal migration and lymphocyte contacts); blue, B-cell area (intranodal migration and lymphocyte contacts); grey, medulla (exit).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Models of immunological synapses and a kinapse. (A)The bull's-eye model of an IS comprises three concentric zones. The cSMAC contains concentrated TCRs and PKC, and is where signalling is terminated. The surrounding pSMAC contains LFA-1 and talin. The dSMAC is enriched in the phosphatase CD45, which is excluded from the synapse to maintain optimal signalling. (B) The zones of the multifocal synapse are less-well defined. The synaptic interface contains many cSMACs, and the LFA-1-rich pSMAC is diffuse. (C) The kinapse represents a migrating cell that forms transient synapses. PKC mediates the transition from a stationary IS to a kinapse, whereas WASp mediates the reverse action. As with both the bull's-eye and multifocal models, signalling microclusters form in the distal area (dSMAC) and move through the pSMAC to the cSMAC, where signalling is terminated. In the polarised cell, the dSMAC corresponds to the lamellipodium, the pSMAC to the mid-region and the cSMAC to the uropod.

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