Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1. (1/165)

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.  (+info)

Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking. (2/165)

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.  (+info)

Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases. (3/165)

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.  (+info)

A large family of endosome-localized proteins related to sorting nexin 1. (4/165)

Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008-1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70-110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.  (+info)

Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection method. (5/165)

A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants.  (+info)

RGS-PX1, a GAP for GalphaS and sorting nexin in vesicular trafficking. (6/165)

Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a Galpha(s)-specific GAP. The RGS domain of RGS-PX1 specifically interacted with Galpha(s), accelerated its GTP hydrolysis, and attenuated Galpha(s)-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking.  (+info)

The Cdc42 target ACK2 interacts with sorting nexin 9 (SH3PX1) to regulate epidermal growth factor receptor degradation. (7/165)

Activated Cdc42-associated kinase-2 (ACK2) is a non-receptor tyrosine kinase that serves as a specific effector for Cdc42, a Rho family small G-protein. Recently, we have found that ACK2 directly interacts with clathrin heavy chain through a clathrin-binding motif that is conserved in all endocytic adaptor proteins and regulates clathrin assembly, suggesting that ACK2 plays a role in clathrin-coated vesicle endocytosis (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473). Here we report the identification of another binding partner for ACK2 that has previously been implicated in endocytosis, namely the sorting nexin protein SH3PX1 (sorting nexin 9). The interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SH3PX1. Co-immunoprecipitation studies indicate that ACK2, clathrin, and SH3PX1 form a complex in cells. Epidermal growth factor (EGF) stimulated the tyrosine phosphorylation of SH3PX1, whereas co-transfection of ACK2 with SH3PX1 resulted in the constitutive phosphorylation of SH3PX1. However, co-transfection of the kinase-dead mutant ACK2(K158R) with SH3PX1 blocked EGF-induced tyrosine phosphorylation of SH3PX1, indicating that the EGF-stimulated phosphorylation of SH3PX1 is mediated by ACK2. EGF receptor levels were significantly decreased following EGF stimulation of cells co-expressing ACK2 and SH3PX1, thus highlighting a novel role for ACK2, working together with SH3PX1 to promote the degradation of the EGF receptor.  (+info)

The beta-appendages of the four adaptor-protein (AP) complexes: structure and binding properties, and identification of sorting nexin 9 as an accessory protein to AP-2. (8/165)

Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the beta-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the beta-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the beta-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel beta-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.  (+info)

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