Integrin undergoes different activation states by changing its quaternary conformation. The integrin ␤ hybrid domain acts as a lever for the transmission of activation signal. Integrins represent a large family of type I heterodimeric (␣ and ␤ subunits) membrane proteins capable of bidirectional signal transduction serving cell growth, differentiation, and apoptosis (1). The ␤ I-like domain is flanked by the hybrid and PSI 1 (for plexins, semaphorins, and integrins) domains (Fig. 1A) (2, 3). The hybrid domain has been shown to be important in the propagation of the activation signal from one end of the ␤ subunit to the other (2, 4-6). Recently, superimposed structural coordinates of liganded-open ␣ IIb ␤ 3 headpiece with that of unliganded-closed ␣ V ␤ 3 revealed an ϳ10-Å shift and a concomitant rotation of the hybrid domain relative to the last helix of the ␤ I-like domain upon ligand binding (7). mAbs that attenuate this swing-out motion of the integrin ␣ 5 ␤ 1 hybrid domain prevent effective allosteric activation of the ␤ I-like domain (5, 6).Collective observations from electron microscopy images and crystal structures of integrin ␣ IIb ␤ 3 , ␣ V ␤ 3 , and ␣ 5 ␤ 1 suggest that integrin may undergo at least three activation states depicted by different quaternary conformations (3, 7-13). A bent integrin with the hybrid domain in close proximity of the integrin epidermal growth factor (I-EGF) 3 and 4 domains represents the resting state. The extended integrin with the hybrid domain distally separated from I-EGF 3 and 4 but orientated toward the ␣ subunit ␤-propeller depicts a low affinity state, whereas the extended integrin with a swing-out hybrid domain away from the ␣ subunit ␤-propeller represents a high affinity state.The conceptualization of different integrin affinity states also derives from earlier functional studies. Observations were made on integrins having different ligand binding properties under different cellular or extracellular conditions. Resting platelet integrin ␣ IIb ␤ 3 binds immobilized fibrinogen, but binding to soluble fibrinogen, fibronectin, or von Willebrand factor requires platelet activation by agonist (14 -16). Divalent cation Mn 2ϩ activates integrin ␣ 4 ␤ 1 to bind VCAM-1, whereas adhesion to fibronectin-derived CS-1 peptide requires additional activating mAb (17). Real time analysis of integrin ␣ 4 ␤ 1 binding to fluorescent-conjugated ligand mimetic via chemokine receptor activation on leukocytes also suggests integrin acquiring multiple affinity states (18). For the integrin ␣ L ␤ 2 , ligand ICAM-1 exhibits higher affinity for purified ␣ L ␤ 2 from T cells than . Prior exposure of ␣ L ␤ 2 to ICAM-1 increased its binding to . Soluble ICAM (sICAM)-3 binds to ␣ L ␤ 2 with 9-fold lower affinity than sICAM-1 (21). We also reported the requirement of two ␣ L ␤ 2 -activating mAbs, KIM185 and KIM127, for their adhesion to ICAM-3 as compared with ICAM-1 (22). Crystal structures of engineered intermediate affinity ␣ L I-domain (L161C/F299C) and high affinity ␣ L I-domain (K287C/K294C...

Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive immunodeficiency caused by mutations in the ␤2 integrin, CD18, that impair CD11/CD18 heterodimer surface expression and/or function. Absence of functional CD11/ CD18 integrins on leukocytes, particularly neutrophils, leads to their incapacity to adhere to the endothelium and migrate to sites of infection. We studied 3 LAD-1 patients with markedly diminished neutrophil CD18 expression, each of whom had a small population of lymphocytes with normal CD18 expression (CD18 ؉ ). These CD18 ؉ lymphocytes were predominantly cytotoxic T cells, with a memory/effector phenotype. Microsatellite analyses proved patient origin of these cells. Sequencing of T-cell subsets showed that in each patient one CD18 allele had undergone further mutation. Interestingly, all 3 patients were young adults with inflammatory bowel disease. Somatic reversions of inherited mutations in primary T-cell immunodeficiencies are typically associated with milder clinical phenotypes. We hypothesize that these somatic revertant CD18 ؉ cytotoxic T lymphocytes (CTLs) may have altered immune regulation. The discovery of 3 cases of reversion mutations in LAD-1 at one center suggests that this may be a relatively common event in this rare disease. IntroductionSomatic mosaicism due to site-specific reversions of inherited mutations to wild type has received much attention in the last decade. 1-7 Some of these reversions have expanded in the host, leading to speculation about possible survival advantages conferred on the cell population carrying the reverted normal sequence. The events leading to somatic mosaicism as a result of reversion of inherited mutations to normal are in principal different from germ-line mosaicism or somatic mosaicism due to de novo mutation. 7 Pathologic mutations that have reverted to wild type have been considered paradigms of endogenous somatic gene therapy. 5,[8][9][10] Unusual phenotypes have alerted investigators to this sort of mosaicism, such as a milder-thanexpected disease course, progressive improvement of disease, or detection of phenotypically normal and abnormal cells. Several different mechanisms have been suggested to be responsible for these events: intragenic recombination, mitotic gene conversion, second-site mutations, DNA slippage, and site-specific reversion to normal. The "back mutation" or "repair" process is most likely random and may reflect an increased mutation rate in certain disorders or mutational hot spots. 7,8,11 Somatic reversions have been described in epidermolysis bullosa, tyrosinaemia type I, Duchenne muscular dystrophy, Lesch-Nyhan disease, Charcot-Marie-Tooth disease type 1A, and Fanconi anemia. [11][12][13][14] Among the primary immunodeficiencies, reversions in adenosine deaminase deficiency, X-linked severe combined immunodeficiency, and Wiskott-Aldrich syndrome confer selective growth advantage on the corrected cells. 1,2,4,5,9,10 Leukocyte adhesion deficiency type-1 (LAD-1) is an autosomal recessive disease caused by ...

Integrin activation involves global conformational changes as demonstrated by various functional and structural analyses. The integrin ␤ hybrid domain is proposed to be involved in the propagation of this activation signal. Our previous study showed that the integrin ␤ 2 -specific monoclonal antibody 7E4 abrogates monoclonal antibody KIM185-activated but not Mg 2؉ / EGTA-activated leukocyte function-associated antigen-1 (LFA-1; ␣ L ␤ 2 )-mediated adhesion to ICAM-1. Here we investigated the allosteric inhibitory property of 7E4. By using human/mouse chimeras and substitution mutations, the epitope of 7E4 was mapped to Val 407 , located in the mid-region of the ␤ 2 hybrid domain. Two sets of constitutively active LFA-1 variants were used to examine the effect of 7E4 on LFA-1/ICAM-1 binding. 7E4 attenuated the binding of variants that have modifications to regions membrane proximal with respect to the ␤ 2 hybrid domain. In contrast, the inhibitory effect was minimal on variants with alterations in the ␣ L I-and ␤ 2 I-like domains preceding the hybrid domain. Furthermore, 7E4 abrogated LFA-1/ICAM-1 adhesion of phorbol 12-myristate 13-acetate-treated MOLT-4 cells. Our data demonstrate that interaction between the hybrid and I-like domain is critical for the regulation of LFA-1-mediated adhesion.Integrins are key proteins involved in cell-cell and cell-matrix interactions, mediating essential biological processes such as embryogenesis, the immune response, and inflammation (1, 2). They are heterodimeric type I membrane glycoproteins formed by noncovalent association of an ␣ and a ␤ subunit. In human, 9 of the 18 ␣ subunits have an I (Inserted)-domain found between blades 2 and 3 of a seven-bladed ␤-propeller structure at its N-terminal end. For this particular subset of integrins, the I-domains are involved in ligand binding via their MIDAS 1 motifs (3-6). The ␤ subunit is linearly organized into an N-terminal PSI-domain (for Plexins, Semaphorins, and Integrins) (7), a spacer region, an I-domain like structure (also known as the I-like domain or ␤ A-domain), a mid-region, a cysteine-rich region containing four tandem IEGF (Integrin Epidermal Growth Factor)-domains, and a terminal domain followed by the transmembrane and cytoplasmic segment (Fig. 1A). In the integrins without an I-domain in their ␣ subunits, the ␤ I-like domain participates directly in ligand binding (8).However, in the integrins with an I-domain in their ␣ subunits, the ␤ I-like domain may, in addition, serve a regulatory role in the integrin-mediated adhesion (9). Previously, we constructed integrin ␤ 2 /␤ 7 chimeras in which the N-terminal region (NTR, the combined PSI domain and spacer segment), I-like domain, and the mid-region of the ␤ 2 subunit were replaced with those of the ␤ 7 subunit (10). The epitopes of five ␤ 2 -specific mAbs were mapped by using these chimeras and were found to require both NTR and mid-region for their expression. Coupled with the observation that removal of the I-like domain did not affect the folding of the NTR-midre...