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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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Co-overexpression of IPIP27 with OCRL1 ΔPIP2 alters endosomal morphology and receptor distribution. CD8-CIMPR HeLaM cells expressing GFP-OCRL1 ΔPIP2 (green) without or with coexpression of mCherry wild-type (WT) IPIP27A or the F224A mutant (red) were labeled with antibodies to TfR (blue) (A) or CD8 (CD8-CIMPR, blue) (B). Bars = 10 μm.
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Figure 10: Co-overexpression of IPIP27 with OCRL1 ΔPIP2 alters endosomal morphology and receptor distribution. CD8-CIMPR HeLaM cells expressing GFP-OCRL1 ΔPIP2 (green) without or with coexpression of mCherry wild-type (WT) IPIP27A or the F224A mutant (red) were labeled with antibodies to TfR (blue) (A) or CD8 (CD8-CIMPR, blue) (B). Bars = 10 μm.

Mentions: To address the functional importance of the interaction between IPIP27 and OCRL1/Inpp5b, we adopted an overexpression strategy. Wild-type and mutant forms of IPIP27 were coexpressed in cells with OCRL1 lacking the entire 5-phosphatase domain (ΔPIP2) or containing a point mutation in the 5-phosphatase domain that renders it catalytically inactive (D499A) (Jefferson and Majerus, 1996), and effects on endosome morphology and receptor distribution were analyzed. The OCRL1 ΔPIP2 mutant acts as a dominant negative in its own right, causing endosome enlargement and redistribution of the TfR and CIMPR to the aberrant endosomes, as described in earlier studies (Choudhury et al., 2005; Hyvola et al., 2006) (Figure 10, A and B). The effects on both endosomal morphology and receptor protein distribution, however, are greatly exacerbated by coexpression with IPIP27A. To confirm that IPIP27 binding to OCRL1 is required to induce these effects, IPIP27A F224A was coexpressed with OCRL1 ΔPIP2. As shown in Figure 10, A and B, the effect of coexpressing IPIP27 with OCRL1 ΔPIP2 on endosome morphology and receptor distribution is abolished by the F224A mutation. Similar results were observed with IPIP27B (unpublished data). In contrast to the ΔPIP2 mutant, OCRL1 D499A alone had little effect on endosome morphology and receptor distribution (Supplemental Figure 6). Coexpression with IPIP27A again caused endosome enlargement, albeit to a lesser degree than with the OCRL1 ΔPIP2 mutant, and there was redistribution of both TfR and CIMPR to the aberrant endosomal compartments, consistent with a defect in their trafficking (Supplemental Figure 6). Again, these effects were abolished by the F224A mutation in IPIP27A, and similar results were seen with IPIP27B (Supplemental Figure 6 and unpublished data). Together, these results strongly suggest that the interaction between IPIP27 and OCRL1 is important for endosomal morphology and function.


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)

Co-overexpression of IPIP27 with OCRL1 ΔPIP2 alters endosomal morphology and receptor distribution. CD8-CIMPR HeLaM cells expressing GFP-OCRL1 ΔPIP2 (green) without or with coexpression of mCherry wild-type (WT) IPIP27A or the F224A mutant (red) were labeled with antibodies to TfR (blue) (A) or CD8 (CD8-CIMPR, blue) (B). Bars = 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 10: Co-overexpression of IPIP27 with OCRL1 ΔPIP2 alters endosomal morphology and receptor distribution. CD8-CIMPR HeLaM cells expressing GFP-OCRL1 ΔPIP2 (green) without or with coexpression of mCherry wild-type (WT) IPIP27A or the F224A mutant (red) were labeled with antibodies to TfR (blue) (A) or CD8 (CD8-CIMPR, blue) (B). Bars = 10 μm.
Mentions: To address the functional importance of the interaction between IPIP27 and OCRL1/Inpp5b, we adopted an overexpression strategy. Wild-type and mutant forms of IPIP27 were coexpressed in cells with OCRL1 lacking the entire 5-phosphatase domain (ΔPIP2) or containing a point mutation in the 5-phosphatase domain that renders it catalytically inactive (D499A) (Jefferson and Majerus, 1996), and effects on endosome morphology and receptor distribution were analyzed. The OCRL1 ΔPIP2 mutant acts as a dominant negative in its own right, causing endosome enlargement and redistribution of the TfR and CIMPR to the aberrant endosomes, as described in earlier studies (Choudhury et al., 2005; Hyvola et al., 2006) (Figure 10, A and B). The effects on both endosomal morphology and receptor protein distribution, however, are greatly exacerbated by coexpression with IPIP27A. To confirm that IPIP27 binding to OCRL1 is required to induce these effects, IPIP27A F224A was coexpressed with OCRL1 ΔPIP2. As shown in Figure 10, A and B, the effect of coexpressing IPIP27 with OCRL1 ΔPIP2 on endosome morphology and receptor distribution is abolished by the F224A mutation. Similar results were observed with IPIP27B (unpublished data). In contrast to the ΔPIP2 mutant, OCRL1 D499A alone had little effect on endosome morphology and receptor distribution (Supplemental Figure 6). Coexpression with IPIP27A again caused endosome enlargement, albeit to a lesser degree than with the OCRL1 ΔPIP2 mutant, and there was redistribution of both TfR and CIMPR to the aberrant endosomal compartments, consistent with a defect in their trafficking (Supplemental Figure 6). Again, these effects were abolished by the F224A mutation in IPIP27A, and similar results were seen with IPIP27B (Supplemental Figure 6 and unpublished data). Together, these results strongly suggest that the interaction between IPIP27 and OCRL1 is important for endosomal morphology and function.

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