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Circular RNA (circRNA) is a class of endogenous non-coding RNAs without 5′ and 3′ ends; an increasing number of studies show that circRNA is involved in skeletal muscle development. From our previous sequencing data, the circRNAome in breast muscle of two chicken lines with a distinct rate of muscle development, which included a fast muscle growing broiler (FMGB) and a slow muscle growing layer (SMGL), we found a novel differentially expressed circRNA generated by intersectin 2 (ITSN2) gene (named circITSN2). We verified that circITSN2 is a skeletal muscle-enriched circRNA that promotes chicken primary myoblast (CPM) proliferation and differentiation. Further molecular mechanism analysis of circITSN2 in chicken myogenesis was performed, and we found circITSN2 directly targeting miR-218-5p. Besides, miR-218-5p inhibits CPM proliferation and differentiation, which is contrary to circITSN2. Commonly, circRNAs act as a miRNA sponge to alleviate the inhibition of miRNAs on mRNAs. Thus, we also identified that a downstream gene LIM domain 7 (LMO7) was inhibited by miR-218-5p, while circITSN2 could block the inhibitory effect of miR-218-5p by targeting it. Functional analysis revealed that LMO7 also accelerates CPM proliferation and differentiation, which was similar to circITSN2 but contrary to miR-218-5p. Taken together, these results suggested that circITSN2 promotes chicken embryonic skeletal muscle development via relieving the inhibition of miR-218-5p on LMO7. Our findings revealed a novel circITSN2/miR-218-5p/LMO7 axis in chicken embryonic skeletal muscle development, which expands our understanding of the complex muscle development regulatory network.

Replicative capacity of normal human cells decreases as telomeric sequence is lost at each division. It is believed that when a subset of chromosomes reach a critically short length, an ATM-initiated and p53-mediated transcriptional response inhibits cell growth, promoting cell senescence. In addition to loss of telomeric sequence, senescence can be induced by other stresses including ionizing radiation, oxidative damage, chemical crosslinkers like the chemotherapeutic agent cisplatin, as well as overactivation of oncogenes and tumor suppressors. Our group found that the expression of an isoform of the INhibitor of Growth 1 gene called ING1a increases approximately 10-fold as fibroblasts approach senescence and that forced expression rapidly induces a senescent phenotype in primary diploid fibroblasts, epithelial and endothelial cells that resembles replicative senescence by most physical and biochemical measures. ING1a induces these changes through strongly inhibiting endocytosis to block mitogen signaling by inducing the expression of intersectin 2, a key scaffolding protein of the endosomal pathway. This, in turn increases the expression of Rb and of p57Kip2 and p16INK4a that serve to maintain Rb is an active, growth inhibitory state. The ING1a model is currently being used to better understand the mechanism(s) responsible for activating Rb to enforce the senescent state.

The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na(+) ) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na(+) regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23∼24∼27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na(+) transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na(+) transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na(+) transport, and implicate miR-27 in aldosterone's action via a novel target. This article is protected by copyright. All rights reserved.

Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42 which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localized at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signalling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N-terminus of RhoU mediated interactions with SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1 increased transferrin accumulation in early endosomes resulting from defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles suggesting that RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

Lung cancer (LC), with its high morbidity and mortality rates, is one of the most widespread and malignant neoplasms. Mediastinal lymph node metastasis (MLNM) severely affects postoperative survival of patients with LC. Additionally, the molecular mechanisms of LC with MLNM (MM LC) remain not well understood. To identify the key biomarkers in its carcinogenesis and development, the datasets GSE23822 and GSE13213 were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and the Database for Annotation, Visualization and Integrated Discovery was used to perform functional annotations of DEGs. Search Tool for the Retrieval of Interacting Genes and Cytoscape were utilized to obtain the protein-protein interaction (PPI) network, and to analyze the most significant module. Subsequently, a Kaplan-Meier plotter was used to analyze overall survival (OS). Additionally, one co-expression network of the hub genes was obtained from cBioPortal. A total of 308 DEGs were identified in the two microarray datasets, which were mainly enriched during cellular processes, including the Gene Ontology terms 'cell', 'catalytic activity', 'molecular function regulator', 'signal transducer activity' and 'binding'. The PPI network was composed of 315 edges and 167 nodes. Its significant module had 11 hub genes, and high expression of actin β, MYC, arginine vasopressin, vesicle associated membrane protein 2 and integrin subunit β1, and low expression of NOTCH1, synaptojanin 2 and intersectin 2 were significantly associated with poor OS. In summary, hub genes and DEGs presented in the present study may help identify underlying targets for diagnostic and therapeutic methods for MM LC.

Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination. These defects were found, at least in part, to be intrinsic to the B cell compartment. In vivo, ITSN2 deficient B cells show a reduction in the expression of SLAM, CD84 or ICOSL that correlates with a diminished ability to form long term conjugates with T cells, to proliferate in vivo, and to differentiate into germinal centre cells. In conclusion, our study not only revealed a key role for ITSN2 as an important regulator of adaptive immune-response during vaccination and viral infection but it is also likely to contribute to a better understanding of human immune pathologies.

Cell senescence contributes to organismal aging and is induced by telomere erosion and an ensuing DNA damage signal as cells reach the end of their replicative lifespan in vitro or in vivo. Stresses induced by oncogene or tumor suppressor hyperactivation, oxidative stress, ionizing radiation and other DNA damaging agents result in forms of stress induced premature senescence (SIPS) that show similarities to replicative senescence. Since replicative senescence and SIPS occur over many days and many population doublings of the mass cultures of primary cells used to study senescence, the sequence of events that occur downstream of senescence signaling can be challenging to define. Here we compare a new model of ING1a-induced senescence with several other forms of senescence. The ING1a epigenetic regulator synchronously induces senescence in mass cultures several-fold faster than all other agents, taking 24 and 36 hours to activate the Rb/ p16INK4a, but not the p53 tumor suppressor axis to efficiently induce senescence. ING1a induces expression of intersectin 2, a scaffold protein necessary for endocytosis, altering the stoichiometry of endocytosis proteins, subsequently blocking growth factor uptake leading to activation of Rb signaling to block cell growth. ING1a acts as a novel link in the activation of the Rb pathway that can impose senescence in the absence of activating p53-mediated DNA damage signaling, and should prove useful in defining the molecular events contributing to Rb-induced senescence.

Intersectins (ITSNs), an evolutionarily conserved adaptor protein family, have been implicated in multiple biologic processes; however, their functions in mammalian oocytes have not been addressed. Here, we report delayed meiotic resumption and defective cytokinesis upon specific depletion of ITSN2 in mouse oocytes. In particular, abnormal spindle, misaligned chromosomes, and loss of cortical actin cap are readily observed in ITSN2-depleted oocytes. Similarly, a small molecule that targets the Cdc42-ITSN interaction also disrupts oocyte maturation and actin polymerization. Moreover, we find that ITSN2 depletion reduces the activity of Cdc42 in oocytes and, of note, that forced expression of the dominant-positive mutant of Cdc42, in part, prevents the effects of ITSN2 knockdown on actin cap formation. In addition, the localization of WASP and Arp2, the downstream effector proteins of Cdc42, is altered in ITSN2-depleted oocytes accordingly. In summary, our data support a model in which ITSN2 depletion induces the inactivation of Cdc42, which, in turn, influences the distribution and function of Arp2/3 and WASP, consequently disrupting oocyte polarity establishment and meiotic division.-Zhang, J., Ma, R., Li, L., Wang, L., Hou, X., Han, L., Ge, J., Li, M., Wang, Q. Intersectin 2 controls actin cap formation and meiotic division in mouse oocytes through the Cdc42 pathway.

Participates in actin remodeling through Rac and receptor endocytosisvia Rab5. Here, we used yeast two-hybrid systemwith Eps8 as bait to screen a human brain cDNA library.ITSN2 was identified as the novel binding factor of Eps8. Theinteraction between ITSN2 and Eps8 was demonstrated by thein vivo co-immunoprecipitation and colocalization assays andthe in vitro GST pull-down assays. Furthermore, we mappedthe interaction domains to the region between amino acids260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition,protein stability assays and immunofluorescence analysisshowed ITSN2 overexpression induced the degradation ofEps8 proteins, which was markedly alleviated with the lysosomeinhibitor NH4Cl treatment. Taken together, our resultssuggested ITSN2 interacts with Eps8 and stimulates the degradationof Eps8 proteins. [BMB reports 2012; 45(3): 183-188]

Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (ΔDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

It is well known that ligand binding to the high-affinity GM-CSF receptor (GMR) activates JAK2. However, how and where this event occurs in a cellular environment remains unclear. Here, we demonstrate that clathrin- but not lipid raft-mediated endocytosis is crucial for GMR signaling. Knockdown expression of clathrin heavy chain or intersectin 2 (ITSN2) attenuated GMR-mediated activation of JAK2, whereas inhibiting clathrin-coated pits or plagues to bud off the membrane by the dominant-negative mutant of dynamin enhanced such event. Moreover, unlike the wild-type receptor, an ITSN2-non-binding mutant of GMR defective in targeting to clathrin-coated pits or plagues [collectively referred to as clathrin-coated structures (CCSs) here] failed to activate JAK2 at such locations. Additional experiments demonstrate that ligand treatment not only enhanced JAK2/GMR association at CCSs, but also induced a conformational change of JAK2 which is required for JAK2 to be activated by CCS-localized CK2. Interestingly, ligand-independent activation of the oncogenic mutant of JAK2 (JAK2V617F) also requires the targeting of this mutant to CCSs. But JAK2V617F seems to be constitutively in an open conformation for CK2 activation. Together, this study reveals a novel functional role of CCSs in GMR signaling and the oncogenesis of JAK2V617F.

Intersectin 2 (ITSN2) is an evolutionarily conserved scaffold protein involved in endocytic internalization, regulation of actin cytoskeleton and epithelial morphogenesis. Recent studies of different Itsn-deficient organisms revealed that this gene is essential for the functioning of the nervous system and for organism viability. Here we report investigations on a possible role of the ITSN2 long isoform in the early embryonic development of Xenopus laevis. In vertebrates, alternative splicing generates several alternatively spliced isoforms of ITSN2. To date the long splice variant of ITSN2 (ITSN2-L) has been reported only for mammals. We show that transcripts of ITSN2-L can be detected in Xenopus embryos from the first cleavage onwards. Overexpression of functional domains of ITSN2-L in embryos resulted in aberrant phenotypes. The strongest phenotype was produced by the C-terminal extension of ITSN2-L. Embryos displayed hyperpigmentation and gastrulation failure that were incompatible with survival. The C-terminus of ITSN2-L includes the DH–PH tandem, a nucleotide exchange factor for the small GTPase Cdc42 and the C2 domain. Further investigations revealed that the DH–PH tandem was responsible for the development of the phenotype affecting the actin cytoskeleton in embryos. Observed developmental defects depended on Cdc42. The effect of expression of the constitutively active GTPase strongly resembled that of the DH–PH tandem. The dominant negative Cdc42 partially rescued developmental defects induced by the expression of the DH–PH tandem. Thus, our data indicate that the ITSN2 exchange factor regulates the activity of Cdc42 during embryo development affecting actin cytoskeleton in Xenopus embryos.

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8–CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8–CD9–CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8–INS2–CD49d–clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.

Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin–Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects., This work was supported by grants from the Human Frontier Science Program (HFSP-CDA 00011/2009) and Marie Curie (IRG-209382) to F. Martín- Belmonte, National Institutes of Health grants (R01 DK067153 and R01 DK074398) to K. Mostov, and grants from the Ministerio de Ciencia e Innovación to F. Martín-Belmonte (BFU2008-01916) and M.A. Alonso (BFU2006- 01925) and to F. Martín-Belmonte and M.A. Alonso (CONSOLIDER CSD2009-00016). An institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also acknowledged.

Background Recent studies have reported large common regions of homozygosity (ROHs) that are the result of autozygosity, that is, the cooccurrence within individuals of long haplotypes that have a high frequency in the population. A recent study reports that such regions are found more commonly in individuals with schizophrenia compared with controls, and identified nine ‘risk ROHs’ that were individually more common in cases. Of these, four contained or neighboured genes associated with schizophrenia (NOS1AP/UHMK1, ATF2, NSF and PIK3C3). Methods We have applied the same methodology to a UK sample of 506 cases with bipolar disorder and 510 controls. Results There was no overall excess of common ROHs among bipolar individuals. With one exception, the haplotypes accounting for the ROHs appeared to be distributed according to the Hardy–Weinberg equilibrium. One ROH was individually more common among cases (uncorrected P = 0.0003). This ROH spanned the chromosome 2p23.3 gene ITSN2 (the gene for intersectin 2 isoform 2). However, inspection of the homozygous haplotypes and haplotype-based tests for association failed to provide a clearer understanding of why this ROH was occurring more commonly. Conclusion Overall, we conclude that, in contrast with schizophrenia, common ROHs are rarely associated with susceptibility to bipolar disorder. This supports the idea that predominantly different genes are increasing susceptibility to schizophrenia and bipolar affective disorders.

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.

Human intersectins 1 and 2 (ITSN1 and ITSN2) are conserved proteins involved in clathrin-mediated endocytosis. In both, two major splice variants, the so-called long and short isoforms have been identified. Whereas most analyses so far focussed on ITSN1, little is known about ITSN2. Data from expression analyses for the intersectin genes mainly refer to the major isoforms. Only recently have a few minor splice variants of ITSN2 been described, though no detailed analyses of their expression in different tissues have been performed. Using RT-PCR-studies we analyzed ITSN2 minor splice variants and their expression in an adult tissue panel. Thereby we demonstrated at least one new minor variant lacking exon 7. Differential expression was demonstrated for a previously described minor splice variant including exon 16 (ITSN2C) with a relative increase in adult human brain tissue. Additional comparative expression analyses in oligodendrogliomas furthermore revealed differential expression with lack of this specific minor splice variant in the brain tumor tissue. These results indicate that ITSN2C may be specifically expressed in neurons hinting to a physiologic role in neuronal cell function.

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-γ stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.

The metastasizing capacity of the rat pancreatic adenocarcinoma BSp73ASML (ASML(wt)) is strikingly reduced by a knockdown of CD44v4-v7 (ASML(kd)). We used this model to analyze the role of the CD44 variant isoform (CD44v) in (pre)metastatic niche formation. Intrafootpad injections of ASML(wt)-, but not ASML(kd)-conditioned medium (CM), strongly promote settlement of ASML(kd) cells in lymph nodes and lung. Fractionation of CM revealed a contribution by a soluble matrix and exosomes, where the CD44v6-containing ASML(wt)-soluble fraction can complement ASML(kd)-exosomes, but not vice versa. This implies that exosomes are the final actors, are CD44v-independent, but require a soluble matrix, which depends on CD44v. Analyzing the composition revealed that only the ASML(wt)-matrix contains c-Met and urokinase-type plasminogen activator receptor. In vitro, mostly ASML(wt)-exosomes promote proliferation and induce gene expression in metastatic organ cells. However, in vivo corresponding changes in the (pre) metastatic organ are only observed when both, exosomes plus the soluble matrix, are provided. Thus, neither CD44v nor exosomes alone suffice for (pre)metastatic niche formation. Instead, CD44v suffices for assembling a soluble matrix, which allows exosomes, independent of their origin from poorly or highly metastatic cells, to modulate (pre) metastatic organ cells for tumor cell embedding and growth.

The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68’s interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.