PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.     

Densin-180 interacts with delta-catenin/neural plakophilin-related armadillo repeat protein at synapses.

Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex.     

MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile alpha motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membrane via the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.     

Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein, which harbors five or six PSD-95/Discs large/ZO-1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and beta-catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S-SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified beta-dystroglycan (beta-DG) as a binding partner for S-SCAM at inhibitory synapses. WW domains of S-SCAM bind to three sequences of beta-DG. We have also revealed that S-SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S-SCAM are involved in the interaction with neuroligin 2. Beta-DG, neuroligin 2 and S-SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti-beta-DG antibody from rat brain. S-SCAM, beta-DG and neuroligin 2 are partially co-localized in rat hippocampal neurons. These data suggest that S-SCAM is associated with beta-DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin-neuroligin complex.     

nRap GEP: a novel neural GDP/GTP exchange protein for rap1 small G protein that interacts with synaptic scaffolding molecule (S-SCAM).

Synaptic scaffolding molecule (S-SCAM) has six PDZ domains through which it interacts with N-methyl-d-aspartate receptors and neuroligin at synaptic junctions. We isolated here a novel S-SCAM-binding protein. This protein has one PDZ, one Ras association, one Ras GDP/GTP exchange protein (Ras GEP) domain, and one C-terminal consensus motif for binding to PDZ domains. We named it nRap GEP (neural Rap GEP). nRap GEP moreover has an incomplete cyclic AMP (cAMP)-binding (CAB) domain. The domain organization of nRap GEP is similar to that of Epac/cAMP-guanine nucleotide exchange factor (GEF) I, except that Epac/cAMP-GEFI has complete CAB and Ras GEP domains but lacks the other two domains and the C-terminal motif. nRap GEP showed GEP activity for Rap1 but did not bind cAMP. nRap GEP was specifically expressed in rat brain. Immunohistochemical analysis revealed that nRap GEP and S-SCAM were localized at synaptic areas of the cerebellum. These results suggest that nRap GEP is a novel neural Rap1-specific GEP which is associated with S-SCAM.     

Synaptic scaffolding molecule interacts with axin

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.     

Association of membrane-associated guanylate kinase-interacting protein-1 with Raf-1

Membrane-associated guanylate kinase-interacting protein (MAGUIN)-1 was identified as a protein interacting with synaptic scaffolding molecule (S-SCAM) and postsynaptic density (PSD)-95/synapse-associated protein (SAP)90. MAGUIN-1 has a chimerical molecular structure composed of one sterile alpha motif, one PSD-95/Dlg-A/ZO-1 (PDZ), and one pleckstrin homology (PH) domain, and interacts with the PDZ domains of S-SCAM and PSD-95/SAP90 via its carboxyl-terminal PDZ-binding motif. MAGUIN-1 is considered as a mammalian homologue of Drosophila CNK, which is a Raf-interacting protein implicated in the regulation of eye development. Here we have tested whether MAGUIN-1 interacts directly with Raf-1. MAGUIN-1 and Raf-1 were coimmunoprecipitated from rat brain. MAGUIN-1 binds to the kinase domain of Raf-1, and Raf-1 binds to the middle region of MAGUIN-1 containing the PH domain. However, in contrast to the dominant active mutant of Ki-Ras, which interacts with Raf-1, recruits it to the plasma membrane from the cytosol, and activates it, MAGUIN-1 neither activates Raf-1 nor recruits it to the plasma membrane. MAGUIN-1 may link Raf-1 to components of synapses assembled by PSD-95/SAP90 and S-SCAM.     

Three isoforms of synaptic scaffolding molecule and their characterization. Multimerization between the isoforms and their interaction with N-methyl-D-aspartate receptors and SAP90/PSD-95-associated protein

The synaptic scaffolding molecule (S-SCAM) has been identified as a protein interacting with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/hDLG-associated protein). S-SCAM has six PDZ (we have numbered them PDZ-0 to -5), two WW, and one guanylate kinase (GK) domains and interacts with N-methyl-D-aspartate (NMDA) receptor via PDZ-5 and SAPAP via the GK domain. We have identified here shorter isoforms of S-SCAM that start at the 164th or 224th methionine, and we renamed the original one, S-SCAMalpha, the middle one, S-SCAMbeta, and the shortest one, S-SCAM-gamma. S-SCAMbeta and -gamma have five PDZ (PDZ-1 to -5), two WW, and one GK domains. S-SCAMalpha interacted with S-SCAMbeta and -gamma through the region containing PDZ-4 and -5. The region containing both of PDZ-4 and -5 is sufficient for the clustering of NMDA receptors and forms a dimer in gel filtration, suggesting that S-SCAM forms multimers via the interaction between the C-terminal PDZ domains and assembles NMDA receptors into clusters. S-SCAMbeta and -gamma also interacted with SAPAP, suggesting that the N-terminal region of the GK domain is not necessary for the interaction. Finally, we have identified the interaction of the PDZ domains of S-SCAM with the GK domain of PSD-95/SAP90. S-SCAM, PSD-95/SAP90, and SAPAP are colocalized at least in some part in brain. Therefore, S-SCAM, PSD-95/SAP90, and SAPAP may form a complex in vivo.     

Presenilin-1 inhibits delta-catenin-induced cellular branching and promotes delta-catenin processing and turnover

Although delta-catenin/neural plakophilin-related armadillo protein (NPRAP) was reported to interact with presenilin-1 (PS-1), the effects of PS-1 on delta-catenin have not been established. In this study, we report that overexpression of PS-1 inhibits the delta-catenin-induced dendrite-like morphological changes in NIH 3T3 cells and promotes delta-catenin processing and turnover. The effects of PS-1 on endogenous delta-catenin processing were confirmed in hippocampal neurons overexpressing PS-1, as well as in the transgenic mice expressing the disease-causing mutant PS-1 (M146V). In addition, disease-causing mutant PS-1 (M146V and L286V) enhanced delta-catenin processing, whereas PS-1/gamma-secretase inhibitors could block the formation of processed forms of delta-catenin. Together, our findings suggest that PS-1 can affect delta-catenin-induced morphogenesis possibly through the regulation of its processing and stability.     

The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

PKC regulates the delta2 glutamate receptor interaction with S-SCAM/MAGI-2 protein

Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that delta2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between delta2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.     

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.     

Delta-catenin at the synaptic-adherens junction

Delta-catenin belongs to the p120-catenin (p120(ctn)) protein family, which is characterized by ten, characteristically spaced Armadillo repeats that bind to the juxtamembrane segment of the classical cadherins. Delta-catenin is the only member of this family that is expressed specifically in neurons, where it binds to PDZ domain proteins in the post-synaptic compartment. As a component of both adherens and synaptic junctions, delta-catenin can link the adherens junction to the synapse and, thereby, coordinate synaptic input with changes in the adherens junction. By virtue of its restriction to the post-synaptic area, delta-catenin creates an asymmetric adherens junction in the region of the synapse. The crucial nature of the specialized function of delta-catenin in neurons is demonstrated by a targeted gene mutation, which causes deficits in learning and in synaptic plasticity. Taken together, recent evidence indicates that delta-catenin is a sensor of synaptic activity and implements activity-related morphological changes at the synapse.     

nArgBP2, a novel neural member of ponsin/ArgBP2/vinexin family that interacts with synapse-associated protein 90/postsynaptic density-95-associated protein (SAPAP).

Postsynaptic density (PSD)-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are synaptic membrane-associated guanylate kinases. Both the proteins interact with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/Dlg-associated protein). SAPAP is a protein highly enriched in the PSD fraction and may link PSD-95/SAP90 and S-SCAM to Triton X-100-insoluble structures. We found here a novel SAPAP-interacting protein, which was specifically expressed in neural tissue and was present in the postsynaptic density fraction in brain. This protein had a sorbin homology domain in the N terminus, a zinc finger motif in the middle region, and three src homology (SH) 3 domains in the C terminus and was homologous to the ponsin/ArgBP2/vinexin family proteins. We named this protein nArgBP2 because it was the most homologous to ArgBP2. nArgBP2 is a neural member of a growing family of SH3-containing proteins. nArgBP2 bound to the proline-rich region of SAPAP via its third SH3 domain and was coimmunoprecipitated with SAPAP from the extract of rat brain. Furthermore, nArgBP2 was colocalized with SAPAP at synapses in cerebellum. nArgBP2 bound to not only SAPAP but also vinculin and l-afadin, known to bind to ponsin and vinexin. nArgBP2 may be implicated in the protein network around SAPAP in the PSD.     

Phosphorylation-regulated cleavage of the tumor suppressor PTEN by caspase-3: implications for the control of protein stability and PTEN-protein interactions

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNFalpha-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylation-regulated manner.     

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction

Delta-catenin (delta-catenin) is a neuron-specific catenin, which has been implicated in adhesion and dendritic branching. Moreover, deletions of delta-catenin correlate with the severity of mental retardation in Cri-du-Chat syndrome (CDCS), which may account for 1% of all mentally retarded individuals. Interestingly, delta-catenin was first identified through its interaction with Presenilin-1 (PS1), the molecule most frequently mutated in familial Alzheimer's Disease (FAD). We investigated whether deletion of delta-catenin would be sufficient to cause cognitive dysfunction by generating mice with a targeted mutation of the delta-catenin gene (delta-cat(-/-)). We observed that delta-cat(-/-) animals are viable and have severe impairments in cognitive function. Furthermore, mutant mice display a range of abnormalities in hippocampal short-term and long-term synaptic plasticity. Also, N-cadherin and PSD-95, two proteins that interact with delta-catenin, are significantly reduced in mutant mice. These deficits are severe but specific because delta-cat(-/-) mice display a variety of normal behaviors, exhibit normal baseline synaptic transmission, and have normal levels of the synaptic adherens proteins E-cadherin and beta-catenin. These data reveal a critical role for delta-catenin in brain function and may have important implications for understanding mental retardation syndromes such as Cri-du-Chat and neurodegenerative disorders, such as Alzheimer's disease, that are characterized by cognitive decline.     

Identification of novel NPRAP/δ-catenin-interacting proteins and the direct association of NPRAP with dynamin 2

Neural plakophilin-related armadillo protein (NPRAP or δ-catenin) is a neuronal-specific protein that is best known for its interaction with presenilin 1 (PS1). Interestingly, the hemizygous loss of NPRAP is associated with severe mental retardation in cri du chat syndrome (CDCS), and mutations in PS1 cause an aggressive, early-onset form of Alzheimer's disease. Until recently, studies on the function of NPRAP have focused on its ability to modulate dendritic protrusion elaboration through its binding to cell adhesion and scaffolding molecules. However, mounting evidence indicates that NPRAP participates in intracellular signaling and exists in the nucleus, where it modulates gene expression. This apparent bifunctional nature suggests an elaborate neuronal role, but how NPRAP came to participate in such distinct subcellular events remains a mystery. To gain insight into this pathway, we immunoprecipitated NPRAP from human SH SY5Y cells and identified several novel interacting proteins by mass spectrometry. These included neurofilament alpha-internexin, interferon regulatory protein 2 binding factors, and dynamins 1 and 2. We further validated dynamin 2/NPRAP colocalization and direct interaction in vivo, confirming their bona fide partnership. Interestingly, dynamin 2 has established roles in endocytosis and actin assembly, and both of these processes have the potential to interface with the cell adhesion and intracellular signaling processes that involve NPRAP. Our data provide new avenues for approaching NPRAP biology and suggest a broader role for this protein than previously thought.