Dynamic interaction of CD2 with the GYF and the SH3 domain of compartmentalized effector molecules

Harvard University, Cambridge, Massachusetts, United States
The EMBO Journal (Impact Factor: 10.43). 12/2002; 21(22):5985-95. DOI: 10.1093/emboj/cdf602
Source: PubMed


Intracellular protein interaction domains are essential for eukaryotic signaling. In T cells, the CD2BP2 adaptor binds two membrane-proximal proline-rich motifs in the CD2 cytoplasmic tail via its GYF domain, thereby regulating interleukin-2 production. Here we present the structure of the GYF domain in complex with a CD2 tail peptide. Unlike SH3 domains, which use two surface pockets to accommodate proline residues of ligands, the GYF domain employs phylogenetically conserved hydrophobic residues to create a single interaction surface. NMR analysis shows that the Fyn but not the Lck tyrosine kinase SH3 domain competes with CD2BP2 GYF-domain binding to the same CD2 proline-rich sequence in vitro. To test the in vivo significance of this competition, we used co-immunoprecipitation experiments and found that CD2BP2 is the ligand of the membrane-proximal proline-rich tandem repeat of CD2 in detergent-soluble membrane compartments, but is replaced by Fyn SH3 after CD2 is translocated into lipid rafts upon CD2 ectodomain clustering. This unveils the mechanism of a switch of CD2 function due to an extracellular mitogenic signal.

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Available from: Ronald Kühne, May 13, 2014
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    • "While a scaffolding function of the protein at the T cell membrane is conceivable (though controversial) (Freund et al., 2002; Heinze et al., 2007), a more unifying concept of CD2BP2 function arose from the observation that its GYF domain (amino acids 280– 341 of the protein) confers localization to the spliceosome. "
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    ABSTRACT: Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulation. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
    Full-text · Article · Jun 2015 · Journal of Molecular Cell Biology
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    • "The main helix is tilted away from the sheet, providing space for an array of stacked aromatic side chains which create the binding site for the PPG motif of the peptide ligand (reviewed in Kofler and Freund (2006)). Monovalent interactions between GYF domains and peptide targets have reported dissociation constants of about 200 lM (Freund et al., 2002). The GYF domain is thought to be involved in splicing and splicing-associated processes, immune cell function and antigen presentation (Kofler and Freund, 2006). "
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    ABSTRACT: Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide-protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats - each complementary to a piece of the target peptide - could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.
    Full-text · Article · Aug 2013 · Journal of Structural Biology
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    • "The small interface between the peptides and their protein domain partners facilitates low-affinity weak interactions that are easily formed and disrupted to regulate cellular responses. Indeed cell surface receptors that mediate immune responses are often coupled to intracellular signaling pathways by recognition of modular protein interaction domains that bind a short LM for example, CD2:CD2BP interaction (KD = μM) [54]. "
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    ABSTRACT: The current version of the human immunome network consists of nearly 1400 interactions involving approximately 600 proteins. Intermolecular interactions mediated by proline-rich motifs (PRMs) are observed in many facets of the immune response. The proline-rich regions are known to preferentially adopt a polyproline type II helical conformation, an extended structure that facilitates transient intermolecular interactions such as signal transduction, antigen recognition, cell-cell communication and cytoskeletal organization. The propensity of both the side chain and the backbone carbonyls of the polyproline type II helix to participate in the interface interaction makes it an excellent recognition motif. An advantage of such distinct chemical features is that the interactions can be discriminatory even in the absence of high affinities. Indeed, the immune response is mediated by well-orchestrated low-affinity short-duration intermolecular interactions. The proline-rich regions are predominantly localized in the solvent-exposed regions such as the loops, intrinsically disordered regions, or between domains that constitute the intermolecular interface. Peptide mimics of the PRM have been suggested as potential antagonists of intermolecular interactions. In this paper, we discuss novel PRM-mediated interactions in the human immunome that potentially serve as attractive targets for immunomodulation and drug development for inflammatory and autoimmune pathologies.
    Full-text · Article · May 2012 · International Journal of Peptides
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