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The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway.

Noakes CJ, Lee G, Lowe M - Mol. Biol. Cell (2011)

Bottom Line: The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1.IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN).The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.

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Related in: MedlinePlus

Interaction of OCRL1 and Inpp5b with IPIP27A and B. (A) Schematic view of human IPIP27A and B showing the predicted PH and coiled-coil domains, and the PxxP motifs found in both proteins. (B) Full-length PH domain and C terminus constructs of IPIP27A and B were tested for interaction with full-length OCRL1 and Inpp5b in the yeast two-hybrid system. Interaction results in growth on high selection medium. (C) GFP-tagged full-length PH domain or C terminus of IPIP27A and B were expressed in HeLa cells and tested for interaction with insect cell expressed in full-length, S-tagged OCRL1 and Inpp5b coupled to beads. Bound proteins were detected by Western blotting with anti-GFP antibodies. (D) Fragments of OCRL1 and Inpp5b were tested for interaction with full-length IPIP27A in the yeast two-hybrid system. (E) Endogenous OCRL1, IPIP27A, and IPIP27B were immunoprecipitated (IP) under native conditions from a HeLa cell extract, and bound proteins were detected by Western blotting with the indicated antibodies. UB, 10% of the unbound fraction. Asterisks indicate background bands that cross-react with the IPIP antibodies. (F) Dimerization of IPIP27 was assessed using extracts from HeLa cells coexpressing GFP- or Myc-tagged IPIP27A and/or IPIP27B followed by native immunoprecipitation with anti-GFP or anti-Myc antibodies and Western blotting with antibodies to these tags.
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Figure 1: Interaction of OCRL1 and Inpp5b with IPIP27A and B. (A) Schematic view of human IPIP27A and B showing the predicted PH and coiled-coil domains, and the PxxP motifs found in both proteins. (B) Full-length PH domain and C terminus constructs of IPIP27A and B were tested for interaction with full-length OCRL1 and Inpp5b in the yeast two-hybrid system. Interaction results in growth on high selection medium. (C) GFP-tagged full-length PH domain or C terminus of IPIP27A and B were expressed in HeLa cells and tested for interaction with insect cell expressed in full-length, S-tagged OCRL1 and Inpp5b coupled to beads. Bound proteins were detected by Western blotting with anti-GFP antibodies. (D) Fragments of OCRL1 and Inpp5b were tested for interaction with full-length IPIP27A in the yeast two-hybrid system. (E) Endogenous OCRL1, IPIP27A, and IPIP27B were immunoprecipitated (IP) under native conditions from a HeLa cell extract, and bound proteins were detected by Western blotting with the indicated antibodies. UB, 10% of the unbound fraction. Asterisks indicate background bands that cross-react with the IPIP antibodies. (F) Dimerization of IPIP27 was assessed using extracts from HeLa cells coexpressing GFP- or Myc-tagged IPIP27A and/or IPIP27B followed by native immunoprecipitation with anti-GFP or anti-Myc antibodies and Western blotting with antibodies to these tags.

Mentions: To determine the cellular role of OCRL1, we searched for new interaction partners. In a genome-wide yeast two-hybrid screen using Drosophila melanogaster proteins, it was reported that the single orthologue of OCRL1 and Inpp5b interacts with a small predicted PH-domain protein, CG12393 (Giot et al., 2003). Whereas insect species have only a single orthologue of CG12393, vertebrates have two versions of this protein, which we name IPIP27A and B (Figure 1A and Supplemental Figure 1). These proteins are the same as Ses1 and 2 recently reported by the De Camilli group (Swan et al., 2010). Both IPIP27A and B interact with OCRL1 and Inpp5b in a directed yeast two-hybrid assay, and binding is mediated by the C-terminal regions of the IPIPs (Figure 1B). These interactions were confirmed using pull-down experiments, with recombinant OCRL1 and Inpp5b efficiently binding to full-length and C-terminal regions of GFP-tagged IPIP27A and B, but not to the PH domains (Figure 1C). The regions of OCRL1 and Inpp5b responsible for binding to the IPIPs were mapped using yeast two-hybrid and pull-down experiments to the C terminus, with both the ASH and RhoGAP-like domains required for efficient binding (Figure 1D). These findings are in agreement with those reported by Swan et al. (2010).


The PH domain proteins IPIP27A and B link OCRL1 to receptor recycling in the endocytic pathway.

Noakes CJ, Lee G, Lowe M - Mol. Biol. Cell (2011)

Interaction of OCRL1 and Inpp5b with IPIP27A and B. (A) Schematic view of human IPIP27A and B showing the predicted PH and coiled-coil domains, and the PxxP motifs found in both proteins. (B) Full-length PH domain and C terminus constructs of IPIP27A and B were tested for interaction with full-length OCRL1 and Inpp5b in the yeast two-hybrid system. Interaction results in growth on high selection medium. (C) GFP-tagged full-length PH domain or C terminus of IPIP27A and B were expressed in HeLa cells and tested for interaction with insect cell expressed in full-length, S-tagged OCRL1 and Inpp5b coupled to beads. Bound proteins were detected by Western blotting with anti-GFP antibodies. (D) Fragments of OCRL1 and Inpp5b were tested for interaction with full-length IPIP27A in the yeast two-hybrid system. (E) Endogenous OCRL1, IPIP27A, and IPIP27B were immunoprecipitated (IP) under native conditions from a HeLa cell extract, and bound proteins were detected by Western blotting with the indicated antibodies. UB, 10% of the unbound fraction. Asterisks indicate background bands that cross-react with the IPIP antibodies. (F) Dimerization of IPIP27 was assessed using extracts from HeLa cells coexpressing GFP- or Myc-tagged IPIP27A and/or IPIP27B followed by native immunoprecipitation with anti-GFP or anti-Myc antibodies and Western blotting with antibodies to these tags.
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Related In: Results  -  Collection

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Figure 1: Interaction of OCRL1 and Inpp5b with IPIP27A and B. (A) Schematic view of human IPIP27A and B showing the predicted PH and coiled-coil domains, and the PxxP motifs found in both proteins. (B) Full-length PH domain and C terminus constructs of IPIP27A and B were tested for interaction with full-length OCRL1 and Inpp5b in the yeast two-hybrid system. Interaction results in growth on high selection medium. (C) GFP-tagged full-length PH domain or C terminus of IPIP27A and B were expressed in HeLa cells and tested for interaction with insect cell expressed in full-length, S-tagged OCRL1 and Inpp5b coupled to beads. Bound proteins were detected by Western blotting with anti-GFP antibodies. (D) Fragments of OCRL1 and Inpp5b were tested for interaction with full-length IPIP27A in the yeast two-hybrid system. (E) Endogenous OCRL1, IPIP27A, and IPIP27B were immunoprecipitated (IP) under native conditions from a HeLa cell extract, and bound proteins were detected by Western blotting with the indicated antibodies. UB, 10% of the unbound fraction. Asterisks indicate background bands that cross-react with the IPIP antibodies. (F) Dimerization of IPIP27 was assessed using extracts from HeLa cells coexpressing GFP- or Myc-tagged IPIP27A and/or IPIP27B followed by native immunoprecipitation with anti-GFP or anti-Myc antibodies and Western blotting with antibodies to these tags.
Mentions: To determine the cellular role of OCRL1, we searched for new interaction partners. In a genome-wide yeast two-hybrid screen using Drosophila melanogaster proteins, it was reported that the single orthologue of OCRL1 and Inpp5b interacts with a small predicted PH-domain protein, CG12393 (Giot et al., 2003). Whereas insect species have only a single orthologue of CG12393, vertebrates have two versions of this protein, which we name IPIP27A and B (Figure 1A and Supplemental Figure 1). These proteins are the same as Ses1 and 2 recently reported by the De Camilli group (Swan et al., 2010). Both IPIP27A and B interact with OCRL1 and Inpp5b in a directed yeast two-hybrid assay, and binding is mediated by the C-terminal regions of the IPIPs (Figure 1B). These interactions were confirmed using pull-down experiments, with recombinant OCRL1 and Inpp5b efficiently binding to full-length and C-terminal regions of GFP-tagged IPIP27A and B, but not to the PH domains (Figure 1C). The regions of OCRL1 and Inpp5b responsible for binding to the IPIPs were mapped using yeast two-hybrid and pull-down experiments to the C terminus, with both the ASH and RhoGAP-like domains required for efficient binding (Figure 1D). These findings are in agreement with those reported by Swan et al. (2010).

Bottom Line: The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1.IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN).The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

ABSTRACT
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely Lowe syndrome (characterized by ocular, nervous system, and renal defects) and type 2 Dent disease (in which only the renal symptoms are evident). The disease mechanisms of these syndromes are poorly understood. Here we identify two novel OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27 kDa (IPIP27)A and B (also known as Ses1 and 2), that also bind the related 5-phosphatase Inpp5b. The IPIPs bind to the C-terminal region of these phosphatases via a conserved motif similar to that found in the signaling protein APPL1. IPIP27A and B, which form homo- and heterodimers, localize to early and recycling endosomes and the trans-Golgi network (TGN). The IPIPs are required for receptor recycling from endosomes, both to the TGN and to the plasma membrane. Our results identify IPIP27A and B as key players in endocytic trafficking and strongly suggest that defects in this process are responsible for the pathology of Lowe syndrome and Dent disease.

Show MeSH
Related in: MedlinePlus