Expression of a structural domain of the β2 subunit essential for αMβ2 ligand recognition

Goodman, Tom; DeGraaf, M.E.; Fischer, H.; Bajt, Mary Lynn

doi:10.1002/jlb.64.6.767

Cited by 8 publications

(9 citation statements)

References 45 publications

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“…CD18 function-blocking mAbs may competitively block integrin-ligand contact, as contact sites recognized by these blocking mAbs lie in the I-like domain of the CD18 subunit that is necessary for ligand binding (14,15). We found that several function-blocking CD18 mAbs prevented the stimulus-induced binding of 327A, 327C, and 330E activation epitope mAbs.…”

Section: Discussionmentioning

confidence: 78%

“…One truncation encoded the amino terminus corresponding to aa 23-457, which consists of the CD18 subunit I-like domain and a critical intrachain disulfide bond (14). A second truncation expressed the cysteine-rich extracellular stalk, residues 411-700.…”

Section: Cd18-blocking Abs Prevent the Induction Of The Activation Epmentioning

confidence: 99%

“…The CD11a subunit of LFA-1 contains an autonomously folding I domain containing a metal-ion binding site critical for ligand binding (12), which is inserted in a predicted ␤-propeller structure also important for ligand recognition (13). The CD18 subunit contains a sequence resembling an I-like domain important for ICAM-1 adhesion, and several function-blocking mAbs recognize this region (14,15). In both subunits, these ligand recognition regions are suspended on a cysteinerich stalk, followed by cytoplasmic tail sequences that influence LFA-1 affinity and clustering (16 -18).…”

mentioning

confidence: 99%

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CD18 Activation Epitopes Induced by Leukocyte Activation

Beals

Edwards

Gottschalk

et al. 2001

The Journal of Immunology

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The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

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Section: Discussionmentioning

confidence: 78%

Section: Cd18-blocking Abs Prevent the Induction Of The Activation Epmentioning

confidence: 99%

mentioning

confidence: 99%

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CD18 Activation Epitopes Induced by Leukocyte Activation

Beals

Edwards

Gottschalk

et al. 2001

The Journal of Immunology

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“…However, because additional sites contributing to ligand binding are predicted in both the ␣L subunit (Stanley et al, 1994) and the ␤2 subunit (Goodman and Bajt, 1996;Goodman et al, 1998), it was possible that an I domaindeleted LFA-1 might bind ligand similarly to a non-I domain-containing integrin. The data in the present study clearly demonstrate that there is no residual ICAM-1 or ICAM-3 ligand binding capacity in ⌬I-LFA-1.…”

Section: Discussionmentioning

confidence: 99%

Effects of I Domain Deletion on the Function of the β2 Integrin Lymphocyte Function-associated Antigen-1

Leitinger

Hogg

2000

MBoC

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A subset of integrin ␣ subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the ␤2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (⌬I-LFA-1) in an LFA-1-deficient T cell line. ⌬I-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), ⌬I-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, ⌬I-LFA-1 had enhanced expression of seven mAb epitopes that are associated with ␤2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, ⌬I-LFA-1 induced constitutive activation of ␣4␤1 and ␣5␤1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on ␤1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of ␤1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.

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“…1) does not directly contribute to ligand binding although it may contain regulatory elements (27). Analogously, the ␤ 3 -and ␤ 2 -subunit C termini have been shown to be unnecessary for ligand binding, since removal of these domains by truncation followed by co-expression with the relevant full-length ␣-subunit generated functional integrins (8,51). Interestingly, both of these ␤-subunit truncations maintained conserved cysteine residues and thereby retained a disulfide knot between the N terminus and mid-section of the ␤-subunit, which was proposed to aid structural integrity (52).…”

mentioning

confidence: 99%

Generation of a Minimal α5β1 Integrin-Fc Fragment

Coe¹,

Askari²,

Kline³

et al. 2001

Journal of Biological Chemistry

109

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The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. Integrins are ␣,␤ heterodimeric transmembrane receptors that play central roles in cell adhesion, migration, differentiation, and survival (1). Several lines of evidence indicate that integrins also contribute to the progression of a wide variety of diseases, including inflammatory, thrombotic, and neoplastic conditions (2-4), and that the integrin families are valid therapeutic targets. The rational design of integrin antagonists based on ligand peptide motifs such as RGD and LDV is currently well advanced. Although the tertiary structure of the integrin heterodimer is unknown, this information would aid the process of drug development, and it represents one of the most important outstanding questions in the field.The overall shape and dimensions of the ␣IIb␤ 3 and ␣ 5 ␤ 1 integrin heterodimers have been revealed by rotary shadowing electron microscopy (5-7). Both receptors consisted of an Nterminal globular head of 8 -12 nm with two extended tails of 18 -20 nm that corresponded to the C termini (7). Similarly, a soluble ␣IIb␤ 3 integrin, generated by removal of the ␣IIb and ␤ 3 transmembrane and cytoplasmic domains, and the ␤ 3 cysteine-rich repeats also contained a globular head, but its tails were 4 -6 nm shorter (8).In the absence of a tertiary structure for the integrin heterodimer, several predictive models have been generated (9 -12), and these have subsequently been supported by biochemical analyses (13)(14)(15)(16). Whereas the ␣-and ␤-subunits are unrelated in primary sequence, they share common structural features including an N-terminal globular ligand-binding domain, C-terminal stalk regions, transmembrane domains, and short cytoplasmic domains (17,18). The N-terminal portion of ␣-subunits contains seven homologous repeats, each 60 -70 amino acid residues in length. These repeats are quite similar in sequence, and repeat four in some integrins and repeats five to seven in all receptors contain EF-hand-like divalent cationbinding motifs. The seven repeats have been predicted to fold cooperatively, forming an all-␤ structure known as a ␤-propeller fold (9) (Fig.

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Expression of a structural domain of the β2 subunit essential for αMβ2 ligand recognition

Cited by 8 publications

References 45 publications

CD18 Activation Epitopes Induced by Leukocyte Activation

CD18 Activation Epitopes Induced by Leukocyte Activation

Effects of I Domain Deletion on the Function of the β2 Integrin Lymphocyte Function-associated Antigen-1

Generation of a Minimal α5β1 Integrin-Fc Fragment

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