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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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Mutational analysis of the IPIP27-OCRL/Inpp5b interaction. (A, top) Sequence alignment of the C terminus of IPIP27A, B and amino acids 403–415 of APPL1. The conserved F and H residues are boxed in red, and the EI residues found only in the IPIPs are boxed in pink. (A, bottom) GFP-tagged IPIP point mutants were expressed in HeLa cells and immunoprecipitated under native conditions using anti-GFP antibodies, and bound OCRL1 was detected by Western blotting. (B) Competition binding assays were performed using the indicated recombinant proteins. A constant amount of S-tagged OCRL1 was coupled to beads, and an equal molar ratio of the minimal OCRL1 binding domain of APPL1 (aa 403–413) was added alone or in the presence of an increasing amount of IPIP27A or B C-terminal constructs (top) or recombinant Rab5 Q79L, Rac1 Q79L, or clathrin terminal domain. Bound proteins were detected by Western blotting with the indicated antibodies. (C) The indicated GFP-tagged Lowe syndrome missense mutants were expressed in HeLa cells, and binding to the indicated GST-tagged bait proteins was monitored by Western blotting. (D) The indicated GFP-tagged OCRL1 missense mutants were expressed in 35S-labeled HeLa cells, immunoprecipitated, and subjected to digestion with increasing amounts of trypsin. Digested proteins were analyzed by SDS–PAGE and autoradiography. G664D is an engineered mutant defective in Rab binding, whereas the others are found in Lowe syndrome patients. The colored asterisks indicate prominent digestion products.
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Figure 2: Mutational analysis of the IPIP27-OCRL/Inpp5b interaction. (A, top) Sequence alignment of the C terminus of IPIP27A, B and amino acids 403–415 of APPL1. The conserved F and H residues are boxed in red, and the EI residues found only in the IPIPs are boxed in pink. (A, bottom) GFP-tagged IPIP point mutants were expressed in HeLa cells and immunoprecipitated under native conditions using anti-GFP antibodies, and bound OCRL1 was detected by Western blotting. (B) Competition binding assays were performed using the indicated recombinant proteins. A constant amount of S-tagged OCRL1 was coupled to beads, and an equal molar ratio of the minimal OCRL1 binding domain of APPL1 (aa 403–413) was added alone or in the presence of an increasing amount of IPIP27A or B C-terminal constructs (top) or recombinant Rab5 Q79L, Rac1 Q79L, or clathrin terminal domain. Bound proteins were detected by Western blotting with the indicated antibodies. (C) The indicated GFP-tagged Lowe syndrome missense mutants were expressed in HeLa cells, and binding to the indicated GST-tagged bait proteins was monitored by Western blotting. (D) The indicated GFP-tagged OCRL1 missense mutants were expressed in 35S-labeled HeLa cells, immunoprecipitated, and subjected to digestion with increasing amounts of trypsin. Digested proteins were analyzed by SDS–PAGE and autoradiography. G664D is an engineered mutant defective in Rab binding, whereas the others are found in Lowe syndrome patients. The colored asterisks indicate prominent digestion products.

Mentions: Swan et al. (2010) identified a conserved C-terminal F&H motif as the OCRL1 binding site in IPIP27/Ses, a motif that is also required for APPL1 binding to OCRL1 (see Supplemental Figure 1 and Figure 2A). As expected, pull-down experiments using truncated versions of IPIP27A and B indicated that binding to OCRL1 and Inpp5b was observed only in the presence of the C-terminal region containing the F&H motif (Supplemental Figure 2). Mutation of either the F or H residues to alanine in IPIP27A or B significantly decreased binding to OCRL1, in agreement with the findings of Swan et al. (2010) (Figure 2A). Mutation of the conserved glutamate–isoleucine (EI) pair of residues also strongly reduced binding to OCRL1 (Figure 2A), consistent with an important role for these residues in conferring high-affinity binding to OCRL1 (Swan et al., 2010). Peptide binding experiments previously suggested that the IPIPs and APPL1 bind to a common binding site in the C-terminal region of OCRL1 (Swan et al., 2010). To confirm this suggestion, we performed competition binding experiments with recombinant proteins. Purified recombinant OCRL1 bound to purified IPIP27A and B, confirming that these proteins interact directly (Figure 2B). Purified OCRL1 also bound to purified APPL1, as observed previously (Erdmann et al., 2007). Addition of a molar excess of IPIP27A or B, however, efficiently competed for this interaction, with binding completely abolished at a 10-fold excess of IPIP27A or B (Figure 2B). In contrast, binding was retained with up to a 50-fold molar excess of Rab5, Rac1, or clathrin heavy-chain terminal domain, that all bind to the ASH-RhoGAP regions of OCRL1, indicating that the competition of APPL1 by IPIPs is specific. These results, together with those of Swan et al. (2010), strongly suggest the IPIPs and APPL1 share a common mode of binding to OCRL1.


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)

Mutational analysis of the IPIP27-OCRL/Inpp5b interaction. (A, top) Sequence alignment of the C terminus of IPIP27A, B and amino acids 403–415 of APPL1. The conserved F and H residues are boxed in red, and the EI residues found only in the IPIPs are boxed in pink. (A, bottom) GFP-tagged IPIP point mutants were expressed in HeLa cells and immunoprecipitated under native conditions using anti-GFP antibodies, and bound OCRL1 was detected by Western blotting. (B) Competition binding assays were performed using the indicated recombinant proteins. A constant amount of S-tagged OCRL1 was coupled to beads, and an equal molar ratio of the minimal OCRL1 binding domain of APPL1 (aa 403–413) was added alone or in the presence of an increasing amount of IPIP27A or B C-terminal constructs (top) or recombinant Rab5 Q79L, Rac1 Q79L, or clathrin terminal domain. Bound proteins were detected by Western blotting with the indicated antibodies. (C) The indicated GFP-tagged Lowe syndrome missense mutants were expressed in HeLa cells, and binding to the indicated GST-tagged bait proteins was monitored by Western blotting. (D) The indicated GFP-tagged OCRL1 missense mutants were expressed in 35S-labeled HeLa cells, immunoprecipitated, and subjected to digestion with increasing amounts of trypsin. Digested proteins were analyzed by SDS–PAGE and autoradiography. G664D is an engineered mutant defective in Rab binding, whereas the others are found in Lowe syndrome patients. The colored asterisks indicate prominent digestion products.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046058&req=5

Figure 2: Mutational analysis of the IPIP27-OCRL/Inpp5b interaction. (A, top) Sequence alignment of the C terminus of IPIP27A, B and amino acids 403–415 of APPL1. The conserved F and H residues are boxed in red, and the EI residues found only in the IPIPs are boxed in pink. (A, bottom) GFP-tagged IPIP point mutants were expressed in HeLa cells and immunoprecipitated under native conditions using anti-GFP antibodies, and bound OCRL1 was detected by Western blotting. (B) Competition binding assays were performed using the indicated recombinant proteins. A constant amount of S-tagged OCRL1 was coupled to beads, and an equal molar ratio of the minimal OCRL1 binding domain of APPL1 (aa 403–413) was added alone or in the presence of an increasing amount of IPIP27A or B C-terminal constructs (top) or recombinant Rab5 Q79L, Rac1 Q79L, or clathrin terminal domain. Bound proteins were detected by Western blotting with the indicated antibodies. (C) The indicated GFP-tagged Lowe syndrome missense mutants were expressed in HeLa cells, and binding to the indicated GST-tagged bait proteins was monitored by Western blotting. (D) The indicated GFP-tagged OCRL1 missense mutants were expressed in 35S-labeled HeLa cells, immunoprecipitated, and subjected to digestion with increasing amounts of trypsin. Digested proteins were analyzed by SDS–PAGE and autoradiography. G664D is an engineered mutant defective in Rab binding, whereas the others are found in Lowe syndrome patients. The colored asterisks indicate prominent digestion products.
Mentions: Swan et al. (2010) identified a conserved C-terminal F&H motif as the OCRL1 binding site in IPIP27/Ses, a motif that is also required for APPL1 binding to OCRL1 (see Supplemental Figure 1 and Figure 2A). As expected, pull-down experiments using truncated versions of IPIP27A and B indicated that binding to OCRL1 and Inpp5b was observed only in the presence of the C-terminal region containing the F&H motif (Supplemental Figure 2). Mutation of either the F or H residues to alanine in IPIP27A or B significantly decreased binding to OCRL1, in agreement with the findings of Swan et al. (2010) (Figure 2A). Mutation of the conserved glutamate–isoleucine (EI) pair of residues also strongly reduced binding to OCRL1 (Figure 2A), consistent with an important role for these residues in conferring high-affinity binding to OCRL1 (Swan et al., 2010). Peptide binding experiments previously suggested that the IPIPs and APPL1 bind to a common binding site in the C-terminal region of OCRL1 (Swan et al., 2010). To confirm this suggestion, we performed competition binding experiments with recombinant proteins. Purified recombinant OCRL1 bound to purified IPIP27A and B, confirming that these proteins interact directly (Figure 2B). Purified OCRL1 also bound to purified APPL1, as observed previously (Erdmann et al., 2007). Addition of a molar excess of IPIP27A or B, however, efficiently competed for this interaction, with binding completely abolished at a 10-fold excess of IPIP27A or B (Figure 2B). In contrast, binding was retained with up to a 50-fold molar excess of Rab5, Rac1, or clathrin heavy-chain terminal domain, that all bind to the ASH-RhoGAP regions of OCRL1, indicating that the competition of APPL1 by IPIPs is specific. These results, together with those of Swan et al. (2010), strongly suggest the IPIPs and APPL1 share a common mode of binding to OCRL1.

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