The role of the cysteine‐rich region of the β2 integrin subunit in the leukocyte function‐associated antigen‐1 (LFA‐1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

Douglass, Wendy; Hyland, Robert H.; Buckley, Christopher D.; Al‐Shamkhani, Aymen; Shaw, Jacqueline; Scarth, Sarah L.; Simmons, David L.; Law, S.K. Alex

doi:10.1016/s0014-5793(98)01498-7

Cited by 31 publications

(44 citation statements)

References 54 publications

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“…In this work, we determined whether 7E4 can block the adhesion of several "constitutively active" LFA-1 to ICAM-1. ␤ 2 NV1 is a ␤ 2 /␤ 1 chimera in which the cysteine-rich region of the ␤ 2 subunit was replaced that that of ␤ 1 (23). ␤ 2 R593C is a mutant found in an LAD-1 patient that supported expression of a constitutively active LFA-1 in an in vitro transfection system (20).…”

Section: Resultsmentioning

confidence: 99%

The Integrin αLβ2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain

Tng

Tan

Ranganathan

et al. 2004

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

The Integrin αLβ2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain

Tng

Tan

Ranganathan

et al. 2004

Journal of Biological Chemistry

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“…These substitutions exposed the CBR LFA-1/2 epitope (53), which we also map within cysteine-rich repeat 3, to residues 534 -546. In other studies, activation of ␣ L ␤ 2 expressed on COS cells was induced if the C-terminal cysteine-rich-repeat region of the ␤ 2 subunit was replaced by that of ␤ 1 (54). A point mutation that introduces a N-glycosylation site into the beginning of cysteine-rich repeat 4 of the ␤ 3 subunit activates integrins ␣ IIb ␤ 3 and ␣ V ␤ 3 (55).…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Antibodies to the C-Terminal Region of the Integrin β2 Subunit Reveals Regions that Become Exposed Upon Receptor Activation

Ferzly

Takagi

et al. 2001

The Journal of Immunology

161

236

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The cysteine-rich repeats in the stalk region of integrin β subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the β2 subunit, one, KIM127, preferentially bound to αLβ2 that was activated by mutations in the cytoplasmic domains, and by Mn2+. KIM127 also bound preferentially to the free β2 subunit compared with resting αLβ2. Activating β2 mutations also greatly enhanced binding of KIM127 to integrins αMβ2 and αXβ2. Thus, the KIM127 epitope is shielded by the α subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated αLβ2 and bound equally well to resting and activated αLβ2, differentially recognized resting αMβ2 and αXβ2, and bound fully to activated αMβ2 and αXβ2. The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344–432 and 432–487, respectively. We thus define five different β2 stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.

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“…27 The extracellular stalk region of CD18 has been shown to be important for effective integrin heterodimer formation, and the cysteinerich repeats near the carboxy-terminal end of the stalk region contain epitopes that define conformational changes critical for activation of alpha subunit binding. 27,28 Two previous studies concluded that the "major cysteine-rich region" is not required for Mac-1 or LFA-1 surface expression. 28,29 However, the patient's mut-1 deletion results in very low levels of surface expression of both LFA-1 and Mac-1, suggesting that specific deletions in this region can alter the CD18 structure sufficiently to cause a profound impairment of integrin heterodimer formation and expression, presumably resulting in intracellular degradation of most monomeric subunits that do not form heterodimers, as reviewed previously.…”

Section: Discussionmentioning

confidence: 99%

“…27,28 Two previous studies concluded that the "major cysteine-rich region" is not required for Mac-1 or LFA-1 surface expression. 28,29 However, the patient's mut-1 deletion results in very low levels of surface expression of both LFA-1 and Mac-1, suggesting that specific deletions in this region can alter the CD18 structure sufficiently to cause a profound impairment of integrin heterodimer formation and expression, presumably resulting in intracellular degradation of most monomeric subunits that do not form heterodimers, as reviewed previously. 1 Northern analysis of CD18 mRNA in transfected COS cells revealed that the low surface expression of Mac-1 and LFA-1 with mut-1 was not accounted for by reduced transcription.…”

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confidence: 99%

Unique CD18 mutations involving a deletion in the extracellular stalk region and a major truncation of the cytoplasmic domain in a patient with leukocyte adhesion deficiency type 1

Hixson

Smith

Shurin

et al. 2004

Blood

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Two novel CD18 mutations were identified in a patient who was a compound heterozygote with type 1 leukocyte adhesion deficiency and whose phenotype was typical except that he exhibited hypertrophic scarring. A deletion of 36 nucleotides in exon 12 (1622del36) predicted the net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the extracellular stalk region (mut-1). A nonsense mutation in exon 15 (2200G>T), predicted a 36-aa truncation of the cytoplasmic domain (mut-2). Lymphocyte functionassociated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) containing the mut-1 ␤ 2 subunit were expressed at very low levels compared with wild-type (wt) ␤ 2 . Mac-1 and LFA-1 expression with the mut-2 ␤ 2 subunit were equivalent to results with wt ␤ 2 . Binding function of Mac-1 with mut-2 ␤ 2 was equivalent to that with wt ␤ 2 . However, binding function of LFA-1 with the mut-2 ␤ 2 subunit was reduced by 50% versus wt ␤ 2 . It was concluded that (1) the portion of the CD18 stalk region deleted in mut-1 is critical for ␤ 2 integrin heterodimer expression but the portion of the cytoplasmic domain truncated in mut-2 is not; and (2) IntroductionImportant insights related to the molecular mechanisms that determine ␤ 2 integrin structure and function have arisen from the study of naturally occurring mutations in the common ␤ 2 subunit of this integrin family. 1,2 A range of mutations in the CD18 gene have been identified in patients with leukocyte adhesion deficiency type 1 (LAD-1), including deletions, truncations, substitutions, frame shifts, and intronic mutations that result in abnormalities of the CD18 protein ranging from absence of protein product, deficient alpha-beta subunit association, and reduced protein size. 1,3-5 All such mutations have resulted in diminished expression of the CD18 integrins on leukocytes, and some mutations also have caused integrin dysfunction. 1,[3][4][5] Patients typically have recurrent bacterial or fungal infections of skin and mucous membranes, impaired mobilization of leukocytes to sites of infection, severe gingivitis/ periodontitis with permanent loss of dentition, and atrophic scarring of wounds. 1,2,6 Laboratory features include a pronounced leukocytosis in the absence of infection, reduced expression of all members of the CD18 or ␤ 2 integrin family on circulating leukocytes, and diminished CD18-dependent leukocyte functions, including cell adhesion, chemotaxis, transendothelial migration, and oxidative burst activation in response to iC3b-coated particles. 1,2,6 We have identified an adult male patient with LAD-1 whose clinical features resemble those of the moderate LAD-1 phenotype, except that his wounds heal with hypertrophic rather than atrophic scarring. His levels of CD18 integrin expression are higher than those of most other reported patients with the moderate phenotype of LAD-1, but the CD18-dependent functions of his leukocytes were similar to those of other moderate phenotype individuals. 1,7 He was found to be a compound heterozygote with 2 novel m...

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The role of the cysteine‐rich region of the β2 integrin subunit in the leukocyte function‐associated antigen‐1 (LFA‐1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

Cited by 31 publications

References 54 publications

The Integrin αLβ2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain

The Integrin αLβ2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain

Epitope Mapping of Antibodies to the C-Terminal Region of the Integrin β2 Subunit Reveals Regions that Become Exposed Upon Receptor Activation

Unique CD18 mutations involving a deletion in the extracellular stalk region and a major truncation of the cytoplasmic domain in a patient with leukocyte adhesion deficiency type 1

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