S.K. Alex Law

Hematologically important mutations: Leukocyte Adhesion Deficiency (first update)

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the β subunit of the β(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed β integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of β integrin conformation.

Two types of transmembrane homomeric interactions in the integrin receptor family are evolutionarily conserved.

Integrins are heterodimers, but recent in vitro and in vivo experiments suggest that they are also able to associate through their transmembrane domains to form homomeric interactions. Two fundamental questions are the biological relevance of these aggregates and their form of interaction in the membrane domain. Although in vitro experiments have shown the involvement of a GxxxG‐like motif, several crosslinking in vivo data are consistent with an almost opposite form of interaction between the transmembrane α‐helices. In the present work, we have explored these two questions using molecular dynamics simulations for all available integrin types. We have tested the hypothesis that homomeric interactions are evolutionary conserved, and essential for the cell, using conservative substitutions to filter out nonnative interactions. Our results show that two models, one involving a GxxxG‐like motif (model I) and an almost opposite form of interaction (model II) are conserved across all α and β integrin types, both in homodimers and homotrimers, with different specificities. No conserved interaction was found for homotetramers. Our results are completely independent from experimental data, both during molecular dynamics simulations and in the selection of the correct models. We rationalize previous seemingly conflicting findings regarding the nature of integrin interhelical homomeric interactions. Proteins 2006.

Transmembrane helices that form two opposite homodimeric interactions : An asparagine scan study of αM and β2 integrins

Integrins are α/β heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG‐like motif, homo‐oligomers observed after in vivo cross‐linking are consistent with an almost opposite helix–helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the α‐helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG‐like motif of the transmembrane domain of α and β integrins, αM and β2. We show that Asn‐mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for αIIb and β3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn‐stabilized dimers were measured by site‐specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane α‐helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin.

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.

Selective recruitment of src family kinase Hck by leukocyte integrin αmβ2 but not αlβ2 or αxβ2

Integrins are type I heterodimeric (α/β) cell adhesion molecules. They trigger cell‐signaling by recruiting cytosolic molecules to their cytoplasmic tails. Integrin α cytoplasmic tail contributes towards integrin function specificity, an important feature of integrins having different α subunits but sharing the same β subunit. Herein, we show that the src family kinase Hck co‐capped selectively with leukocyte integrin αMβ2 but not αLβ2 or αXβ2. This was disrupted when the αM cytoplasmic tail was substituted with that of αL or αX. Co‐capping was recovered by αL or αX cytoplasmic tail truncation or forced separation of the α and β cytoplasmic tails via salt‐bridge disruption.

Integrin CD11a cytoplasmic tail interacts with the CD45 membrane-proximal protein tyrosine phosphatase domain 1

Leucocyte adhesion receptor integrin CD11aCD18 and the transmembrane receptor‐like protein tyrosine phosphatase (RPTP) CD45 mediate immune synapse formation and signalling during antigen presentation. Previous cocapping studies on human naïve T cells demonstrate an interaction between CD11aCD18 and CD45. CD45 cross‐linking also has an effect on the ligand‐binding activity of CD11aCD18. However, the mode of interaction between CD11aCD18 and CD45 remains unclear. Herein, yeast two‐hybrid analysis identified a partial CD45 cytoplasmic tail interacting with that of CD11a. The CD45 cytoplasmic tail comprises a membrane proximal (Mp) region, protein tyrosine phosphatase domain 1 (D1), spacer, D2, and carboxyl terminus. CD45u2003Mp‐D1 was found to be the main interacting region for the CD11a cytoplasmic tail. In contrast, the full‐length CD45 cytoplasmic tail interacted weakly with that of CD11a. It has been reported that CD45 Mp‐D1 but not the full‐length cytoplasmic tail forms a homodimer whose enzymatic activity is inhibited. Our in vitro binding and enzymatic assays showed that the homodimeric CD45 cytoplasmic tail interacts with that of CD11a. The biological function of CD45 dimerization and its association with CD11a remains to be investigated.

Characterization of single amino acid substitutions in the β2 integrin subunit of patients with leukocyte adhesion deficiency (LAD)-1

Leukocyte adhesion deficiency 1 (LAD-1) is caused by defects in the β2 integrin subunit. We studied 18 missense mutations, 14 of which fail to support the surface expression of the β2 integrins. Integrins with the β2-G150D mutation fail to bind ligands, possibly due to the failure of the α1 segment of the βI domain to assume an α-helical structure. Integrins with the β2-G716A mutation are not maintained in their resting states, and the patient has the severe phenotype of LAD-1. The β2-S453N and β2-P648L mutants support the expression of integrins and adhesion functions. They should be re-classified as polymorphic variants.

Function and conformation analyses of an aspartate substitution of the invariant glycine in the integrin βI domain α1-α1′ helix

We showed that the αLβ2 integrin with the non-functional mutation G150D cannot be induced with Mg/EGTA to express the mAb KIM127 epitope, which reports the leg-extended conformation. We extended the study to the αIIbβ3, an integrin without an αI domain. The equivalent mutation, i.e. G161D, also resulted in an expressible, but non-adhesive αIIbβ3 integrin. An NMR study of synthetic peptides spanning the α1-α1′ helix of the β3 I domain shows that both wild-type and mutant peptides are α-helical. However, whereas in the wild-type peptide this helix is continuous, the mutant presents a discontinuity, or kink, precisely at the site of mutation G161D. Our results suggest that the mutation may lock integrin heterodimers in a bent conformation that prevents integrin activation via conformational extension.

The integrin αL leg region controls the Mg/EGTA mediated activation of LFA-1

We have shown that Mg/EGTA (5 mM Mg(2+) and 1.5 mM EGTA) could effectively promote the adhesion of integrin αLβ2 to its ligand ICAM-1 but could not promote that of the αMβ2 to denatured BSA. In order to determine the structural differences between αL and αM that specifically contribute to Mg/EGTA sensitivity, a series of αL/αM chimeras were constructed. Our results showed that αLβ2 with αM calf-1 domain completely lost the response to Mg/EGTA activation. In the reverse experiment, αMβ2 would require the presence of both the αL calf-1 and calf-2 domain to initiate the Mg/EGTA sensitivity.