Crystal Structure and Binding Properties of the CD2 and CD244 (2B4)-binding Protein, CD48

Evans, Edward J.; Castro, Miriam Susana; O’Brien, Rita Cruise; Kearney, Alice; Walsh, Heather L.; Sparks, Lisa M.; Tucknott, Michael G.; Davies, E; Carmo, Alexandre M.; Merwe, P. Anton van der; Stuart, David I.; Jones, E Y; Ladbury, John E.; Ikemizu, Shinji; Davis, Simon J.

doi:10.1074/jbc.m601314200

Cited by 35 publications

(31 citation statements)

References 65 publications

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“…This led to the identification of amino acid residues comprising the Ln511/521-binding site on Lu gp. An unexpected finding is that, unlike most cell-cell interactions that characteristically result from the formation of cell adhesion pairs between the membrane-distal domains of cell adhesion molecules (eg, CD2:CD58 26 and CD2: CD48 27 ), Lu gp uses a more membrane-proximal interdomain junction region to form a critical part of its Ln-binding site.…”

Section: Introductionmentioning

confidence: 94%

The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

Mankelow

Burton

Stefansdottir

et al. 2007

Blood

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The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the ␣5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease. IntroductionThe Lutheran glycoprotein (Lu gp) cellular adhesion molecule is widely expressed in human tissues 1 and is known for carrying antigens of the Lutheran blood group system. On erythrocytes, Lu gp is expressed as 2 isoforms of 78 and 85 kDa. 2 Both share a common extracellular portion that has previously been predicted to comprise 5 immunoglobulin superfamily (IgSF) domains. 1 The 78-kDa isoform (also known as BCAM 3 or Lu[v13] 4 ) results from alternative splicing and lacks 40 C-terminal amino acids within the cytoplasmic domain that contain an SH3-binding motif, a dileucine motif responsible for basolateral targeting, 5 and 5 potential phosphorylation sites. 1 Lu gp binds specifically and with high affinity to the extracellular matrix (ECM) protein Laminin (Ln) containing the ␣5 subunit 6-9 (Laminin 511 and Laminin 521 (Ln511/521) (numbering as in Aumailley et al 10 ). This interaction plays a direct role in the pathophysiology of sickle cell disease by mediating adhesion of sickle cells, via Lu gp, to exposed Ln511/521 of inflamed or damaged vascular endothelium. 9,11,12 Recent studies have shown that higher than normal intracellular levels of cAMP in sickle erythrocytes influence a protein kinase A-mediated or Rap1-mediated signaling pathway resulting in increased adhesion of sickle cells to the basement membrane glycoproteins Ln511/ 521. 13,14 Furthermore, phosphorylation of the 85-kDa Lu gp at S621 in epinephrine-stimulated K562 cells alters adhesion to Ln511/521. 15 The Ln-binding activity of Lu gp is a property of the amino terminal region of Lu gp, and has been localized to a region predicted to form the first 3 IgSF domains (D1D2D3). 6,16 The complementary binding site on Ln is located within a large carboxyl-terminal globular domain of the ␣5 subunit, known to comprise 5 ...

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Section: Introductionmentioning

confidence: 94%

The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

Mankelow

Burton

Stefansdottir

et al. 2007

Blood

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“…CD2 associates with the Src family tyrosine kinases Lck and Fyn and through these kinases couples to downstream signal transduction pathways (18 -20). Proline-rich sequences of the cytoplasmic domain of CD2 have been shown to be involved in the interaction with the Src homology 3 (SH3) 4 domains of these kinases (20 -22), also with the SH3 domain of the adaptor protein CD2AP (23), connecting CD2 to the cytoskeleton, and additionally with the SH3 domain of CD2BP1 (24), resulting in the down-regulation of activation-dependent CD2 adhesion. CD2 binds to the GYF domain of CD2BP2 as well, an interaction resulting in cytokine production (25).…”

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confidence: 99%

“…The extracellular domain of CD2 is composed by two Ig superfamily domains (2), of which the membrane-distal V-like domain is involved in the binding to the ligand (3). CD2 has different ligands in rodents (CD48) and humans (CD58), and the mechanisms of ligand binding are substantially different, as assessed by thermodynamic analysis (4,5). Nevertheless, engagement of CD2 to CD58 in humans, or to CD48 in rodents, facilitates adhesion between T cells and APC and is proposed to promote the formation of an optimal intercellular membrane spacing (ϳ140 Å) suitable for TCR recognition of a peptide Ag bound to MHC (6).…”

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confidence: 99%

Protein Interactions between CD2 and Lck Are Required for the Lipid Raft Distribution of CD2

Nunes

Castro²,

Gonçalves³

et al. 2008

The Journal of Immunology

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In T lymphocytes, lipid rafts are preferred sites for signal transduction initiation and amplification. Many cell membrane receptors, such as the TCR, coreceptors, and accessory molecules associate within these microdomains upon cell activation. However, it is still unclear in most cases whether these receptors interact with rafts through lipid-based amino acid modifications or whether raft insertion is driven by protein-protein interactions. In murine T cells, a significant fraction of CD2 associates with membrane lipid rafts. We have addressed the mechanisms that control the localization of rat CD2 at the plasma membrane, and its redistribution within lipid rafts induced upon activation. Following incubation of rat CD2-expressing cells with radioactive-labeled palmitic acid, or using CD2 mutants with Cys226 and Cys228 replaced by alanine residues, we found no evidence that rat CD2 was subjected to lipid modifications that could favor the translocation to lipid rafts, discarding palmitoylation as the principal mechanism for raft addressing. In contrast, using Jurkat cells expressing different CD2 and Lck mutants, we show that the association of CD2 with the rafts fully correlates with CD2 capacity to bind to Lck. As CD2 physically interacts with both Lck and Fyn, preferentially inside lipid rafts, and reflecting the increase of CD2 in lipid rafts following activation, CD2 can mediate the interaction between the two kinases and the consequent boost in kinase activity in lipid rafts.

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“…The resulting interaction between CD6 and CD166 is therefore unusual, because most other T cell surface proteins mediating cell-cell interactions bind through their N-terminal domains. However, affinity and kinetic measurements revealed that, in solution, binding occurs with a K d of 0.4 -1.0 M (14), relatively strong compared with most other leukocyte adhesion pairs, albeit in the range of low-affinity interactions characteristic of cell surface receptors (25)(26)(27).…”

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confidence: 99%

Extracellular Isoforms of CD6 Generated by Alternative Splicing Regulate Targeting of CD6 to the Immunological Synapse

Castro

Oliveira

Nunes

et al. 2007

The Journal of Immunology

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The great majority of mammalian genes yield multiple transcripts arising from differential mRNA processing, but in very few instances have alternative forms been assigned distinct functional properties. We have cloned and characterized a new isoform of the accessory molecule CD6 that lacks the CD166 binding domain and is expressed in rat and human primary cells. The novel isoform, CD6Δd3, results from exon 5 skipping and consequently lacks the third scavenger receptor cysteine-rich (SRCR) domain of CD6. Differential expression of the SRCR domain 3 resulted in a remarkable functional difference: whereas full-length CD6 targeted to the immunological synapse, CD6Δd3 was unable to localize at the T cell:APC interface during Ag presentation. Analysis of expression of CD6 variants showed that, while being more frequent in coexpression with full-length CD6, the CD6Δd3 isoform constituted the sole species in a small percentage of T cells. In the rat thymus, CD6Δd3 is less represented in double-positive thymocytes but is detectable in nearly 50% of single-positive CD4 or CD8 thymocytes, suggesting that CD6 switching between full-length and Δd3 isoforms may be involved in thymic selection. Strikingly, CD6Δd3 is markedly up-regulated upon activation of T lymphocytes, partially substituting full-length CD6, as evaluated by RT-PCR analysis at the single-cell level, by immunoblotting, and by flow cytometry using Abs recognizing SRCR domains 1 and 3 of human CD6. This elegant mechanism controlling the expression of the CD166 binding domain may help regulate signaling delivered by CD6, through different types of extracellular engagement.

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Crystal Structure and Binding Properties of the CD2 and CD244 (2B4)-binding Protein, CD48

Cited by 35 publications

References 65 publications

The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

Protein Interactions between CD2 and Lck Are Required for the Lipid Raft Distribution of CD2

Extracellular Isoforms of CD6 Generated by Alternative Splicing Regulate Targeting of CD6 to the Immunological Synapse

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