Novel interaction partners of the CD2BP2-GYF domain

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.     

GYF domain proteomics reveals interaction sites in known and novel target proteins

GYF domains are conserved eukaryotic adaptor domains that recognize proline-rich sequences. Although the structure and function of the prototypic GYF domain from the human CD2BP2 protein have been characterized in detail, very little is known about GYF domains from other proteins and species. Here we describe the binding properties of four GYF domains of various origins. Phage display in combination with SPOT analysis revealed the PPG(F/I/L/M/V) motif as a general recognition signature. Based on these results, the proteomes of human, yeast, and Arabidopsis thaliana were searched for potential interaction sites. Binding of several candidate proteins was confirmed by pull-down experiments or yeast two-hybrid analysis. The binding epitope of the GYF domain from the yeast SMY2 protein was mapped by NMR spectroscopy and led to a structural model that accounts for the different binding properties of SMY2-type GYF domains and the CD2BP2-GYF domain.     

Cross-species analyses identify the BNIP-2 and Cdc42GAP homology (BCH) domain as a distinct functional subclass of the CRAL_TRIO/Sec14 superfamily

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ~100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs ('h' is large and hydrophobic residue and 's' is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding 'BCH-only' domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, 'BCH-like' and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases.     

The GYF domain

GYF domains are small, versatile adaptor domains that recognize proline-rich sequences (PRS). They are present in most eukaryotic species sequenced so far, but in contrast to other PRS-recognition domains (PRD), GYF domains have not experienced the same amplification in metazoa during evolution. Mutational and structural analysis has shown the conserved signature W-X-Y-X(6-11)-GPF-X(4)-M-X(2)-W-X(3)-GYF to be the site of interaction with proline-rich peptides. In contrast, composition and length of the C-terminal half of GYF domains are not conserved. Similar to other PRD, GYF domains bind to many different PRS that converge on a minimal consensus sequence. All GYF domains analyzed so far selected for the core motif PPG, whereas amino-acid preferences adjacent to this motif vary. As a result of this analysis, two subfamilies have been identified: CD2BP2-type and SMY2-type GYF domains. The latter subfamily comprises most GYF domains and is characterized by a shorter beta(1)-beta(2) loop and an aspartate instead of the tryptophan found at position 8 in CD2BP2-type GYF domains. Recent analysis of binding specificities for GYF domains allowed identification of novel interaction partners. Thereby proteomics has contributed to a functional understanding of GYF domain-containing proteins and sets the stage for a more systematic investigation of their functions in vivo.     

Alternative binding modes of proline-rich peptides binding to the GYF domain

Recognition of proline-rich sequences plays an important role for the assembly of multiprotein complexes during the course of eukaryotic signal transduction and is mediated by a set of protein folds that share characteristic features. The GYF (glycine-tyrosine-phenylalanine) domain is known as a member of the superfamily of recognition domains for proline-rich sequences. Recent studies on the complexation of the CD2BP2-GYF domain with CD2 peptides showed that the peptide adopts an extended conformation and forms a polyproline type-II helix involving residues Pro4-Pro7 [Freund et al. (2002) EMBO J. 21, 5985-5995]. R/K/GxxPPGxR/K is the key signature for the peptides that bind to the GYF domain [Kofler et al. (2004) J. Biol. Chem. 279, 28292-28297]. In our combined theoretical and experimental study, we show that the peptides adopt a polyproline II helical conformation in the unbound form as well as in the complex. From molecular dynamics simulations, we identify a novel binding mode for the G8W mutant and the wild-type peptide (shifted by one proline in register). In contrast, the conformation of the peptide mutant H9M remains close to the experimentally derived wild-type GYF-peptide complex. Possible functional implications of this altered conformation of the bound ligand are discussed in the light of our experimental and theoretical results.     

Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.     

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

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.     

A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion.

A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment. Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2. In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.     

Regulation of FasL expression: a SH3 domain containing protein family involved in the lysosomal association of FasL

As a death factor of T cells and Natural Killer (NK) cells, Fas Ligand (FasL) is stored in association with secretory lysosomes. Upon stimulation, these cytotoxic granules are transported to the cell membrane where FasL is exposed on the cell surface, shed or secreted. It has been noted before that the proline-rich domain within the cytosolic part of FasL is required for its vesicular association. However, the molecular interactions involved in targeting FasL to secretory lysosomes or to the plasma membrane have not been elucidated. We now identified a family of structurally related proteins that upon co-expression with FasL reallocate the death factor from a membrane to an intracellular localization. Members of this protein family are characterized by a similar domain structure and include FBP17, PACSIN1-3, CD2BP1, CIP4, Rho-GAP C1 and several hypothetical proteins. We show that all tested members of this "FCH/SH3-family" co-precipitate FasL from transfectants. The interactions strictly depend on functional SH3 domains within the FCH/SH3 proteins. Since co-expression of FasL with individual FCH/SH3 proteins dramatically alters the intracellular localization of FasL especially in non-hematopoietic cells, our data suggest that FCH/SH3 proteins might play an important role for the subcellular distribution and lysosomal association of FasL.     

GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.     

Alefacept, an immunomodulatory recombinant LFA-3/IgG1 fusion protein, induces CD16 signaling and CD2/CD16-dependent apoptosis of CD2(+) cells

Alefacept, an immunomodulatory recombinant fusion protein composed of the first extracellular domain of LFA-3 fused to the human IgG1 hinge, C(H)2, and C(H)3 domains, has recently been shown in phase II and III clinical trials to safely reduce disease expression in patients with chronic plaque psoriasis. Alefacept modulates the function of and selectively induces apoptosis of CD2(+) human memory-effector T cells in vivo. We have sought to gain further understanding of the mechanisms of action that influence the biological activity of alefacept and may contribute to its efficacy and patient responsiveness. Specifically evaluated is the ability of alefacept to activate intracellular signals mediated via CD2 and/or Fc gamma RIII (CD16). Experimentation using isoforms of alefacept engineered to have amino acid substitutions in the IgG1 C(H)2 domain that impact Fc gamma R binding indicate that alefacept mediates cognate interactions between cells expressing human CD2 and CD16 to activate cells, e.g., increase extracellular signal-regulated kinase phosphorylation, up-regulate cell surface expression of the activation marker CD25, and induce release of granzyme B. In the systems used, this signaling is shown to require binding to CD2 and CD16 and be mediated through CD16, but not CD2. Experimentation using human CD2-transgenic mice and isoforms of alefacept confirmed the requirement for Fc gamma R binding for detection of the pharmacological effects of alefacept in vivo. Thus alefacept acts as an effector molecule, mediating cognate interactions to activate Fc gamma R(+) cells (e.g., NK cells) to induce apoptosis of sensitive CD2(+) target cells.     

Interaction between the N-terminal SH3 domain of Nck-alpha and CD3-epsilon-derived peptides: non-canonical and canonical recognition motifs

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.     

Single channel function of inositol 1,4,5-trisphosphate receptor type-1 and -2 isoform domain-swap chimeras

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893-3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.     

Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phosphorylation of a common site in CD3epsilon

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3epsilon to Eps8L1 and Nck SH3 domains. CD3epsilon bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3epsilon to interact with either SH3 or SH2 domain-containing proteins.     

A single polymorphism disrupts the killer Ig-like receptor 2DL2/2DL3 D1 domain

Genetic polymorphisms found in the killer Ig-like receptor (KIR), two domains, long cytoplasmic tail 2/3 (KIR2DL2/3) locus are responsible for the differential binding of KIR2DL2/3 allelic products with their HLA-C ligands and have been associated with the resolution of hepatitis C infection. In our study, a KIR CD3zeta fusion-binding assay did not detect any interaction between the KIR2DL2*004 extracellular domain and several putative KIR2DL2/3 ligands. To determine the amino acid polymorphism(s) responsible for the KIR2DL2*004 phenotype, we mutated the polymorphic residues of full-length KIR and expressed them in human Jurkat cells. Flow cytometry analysis failed to detect the surface expression of receptors containing a threonine at position 41 (T41), a polymorphism specific to KIR2DL2*004. Confocal microscopy showed that receptors containing T41 were retained inside the cell and had a perinuclear localization, possibly indicating that their extracellular domain was misfolded. Most KIR2DL2/3 alleles possess an arginine at position 41 (R41), and we predicted through molecular modeling and demonstrated by mutagenesis that R41 most likely interacts with the nearby residues Y77 and D47. Interaction between these residues would maintain C strand contact with the C' and F strands of the D1 domain beta-sheet. Furthermore, R41 and Y77 are conserved in the C and F strand amino acid alignments of Ig-like superfamily members, and may therefore be necessary for the structural integrity of other immune response proteins. Our data indicate that the extracellular T41 polymorphism encoded by the KIR2DL2*004 allele most likely results in misfolding of the D1 domain and complete intracellular retention of the receptor.     

Complete predicted three-dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin superfamily

Tetraspanins are a superfamily of transmembrane proteins implicated in cellular development, motility, and activation through their interactions with a large range of proteins and with specific membrane microdomains. The complete three-dimensional structure of the tetraspanin CD81 has been predicted by molecular modeling and from the crystallographic structure of the EC2 large extracellular domain. Periodicity of sequence conservation, homology modeling, secondary structure prediction, and protein docking were used. The transmembrane domain appears organized as a four-stranded left-handed coiled coil directly connecting to two helices of the EC2. A smaller extracellular loop EC1 contains a small largely hydrophobic beta-strand that packs in a conserved hydrophobic groove of the EC2. The palmitoylable intracellular N-terminal segment forms an amphipathic membrane-parallel helix. Structural variability occurs mainly in an hypervariable subdomain of the EC2 and in intracellular regions. Therefore, the variable interaction selectivity of tetraspanins originates both from sequence variability within structurally conserved domains and from the occurrence of small structurally variable domains. In CD81 and other tetraspanins, the numerous membrane-exposed aromatic residues are asymmetrically clustered and protrude on one side of the transmembrane domain. This may represent a functional specialization of these two sides for interactions with cholesterol, proteins, or membrane microdomains.