A: A 90 kd protein binds OCRL and INPP5B in pulldown experiments. GST and GST fusion proteins of the COOH-terminal regions of OCRL and INPP5B were incubated with rat brain lysate and bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. B: Anti-APPL1 western blotting of the samples shown in A as well as bound proteins from pulldowns in which GST-fusions of full length INPP5B and OCRL were used as baits. C: The interaction between OCRL/INPP5B and APPL1 is direct. His-tagged recombinant APPL1 was incubated with the indicated GST-fusion proteins and bound proteins were analyzed by western-blotting. D: OCRL and INPP5B interact with APPL1 but not with APPL2 as shown by GST-pulldowns from transfected cells followed by western blotting. E: Coprecipitation of OCRL with APPL1 from extracts of transfected Cos-7 cells. Western blotting of the starting lysate and of the immunoprecipitates are shown. F: Epifluorescence images demonstrating that APPL1 colocalizes with OCRL and INPP5B on endosomes in transfected Cos-7 cells. G: H and I: TIRF microscopy sequential images (12 sec intervals) of spots positive for RFP-clathrin, EGF-OCRL and EGFP- or RFP-APPL1 at the cell surface (Bars = F, 2.5 μm; G, H and I = 1μm).

A: Modular structure and interactions of APPL1. The figure also shows an alignment of the 11-mer peptide of human APPL1 with the corresponding sequence of APPL from other species, demonstrating its evolutionary conservation. The sequence is not conserved in human APPL2. Potential phosphorylation sites within the 11-mer peptide are indicated by asterisks. B: GST pulldowns from extracts of Cos-7 expressing either Myc-OCRL or Xpress-INPP5B define an 11 amino acid (residues 403–413) stretch of human APPL1 as the minimal region necessary and sufficient for binding. Bound proteins were identified by western blotting. Numbers indicate the amino acid boundaries of the APPL1 fragment fused to GST. The F404A mutation abolished binding. C: ITC analysis of the binding of the wildtype APPL1 11-mer peptide and of the F404A-mutant peptide. Raw data are shown in the upper panels, and plots of the total heat released as a function of the molar ratio of each ligand are shown in the bottom panels. D: GST-fusions of the wildtype or mutant (S403D and S410D) minimal binding region (11-mer peptide) were incubated with the catalytic subunit of PKA or with type II Ca²⁺-calmodulin dependent kinase (CamKII) in the presence of γ-³²P-ATP. Proteins were separated by SDS-PAGE and the corresponding autoradiography is shown. E: Co-immunoprecipitation of full length wildtype and mutant (S410D) APPL1 with OCRL. F: PKA overexpression impairs binding of APPL1 to OCRL in vivo. For E and F Cos-7 cells were transfected with the indicated expression constructs. Protein complexes were immunoprecipitated using anti-GFP antibody and the starting lysates and the immunoprecipitates were analyzed by western blotting.

A: Close view of the local structural environment of three single amino acid patient mutations in the COOH-terminal regio n of OCRL. B: GST-pulldowns from extracts of rat brain (APPL1, clathrin) or transfected Cos-7 cells (Myc-RacV12) on wildtype and mutant fusion proteins, demonstrating that each of the three mutations abolish binding to APPL1 but not to clathrin or Rac (Myc-RacV12). Bound proteins were detected by western blotting. C: Two colour fluorescence of cells expressing RFP-APPL1 and EGFP-OCRL (wildtype or mutant). Wildtype OCRL is localized on spots throughout the cell. The more peripheral spots (arrowheads), but not all spots (arrows), colocalize with APPL1. Each of three patient mutations abolishes the colocalization with APPL1, although APPL1 positive spots at the periphery persist. Note that at least for the Δ585 mutation, OCRL still has a punctate subcellular distribution.

A: Immunofluorescence of the intracellular distribution of Myc-tagged OCRL in Cos-7 cells and comparison with the distribution of co-transfected Xpress-tagged INPP5B. B: RFP-OCRL (and RFP-INPP5B) colocalizes with EGFP-rab5 positive endosomes but not with EGFP-Rab9-positive organelles. C: EGFP-OCRL positive spots are directly adjacent to endogenous retromer (SNX1) immunoractivity suggesting budding of retromer positive membrane from OCRL positive endosomes. D: Modular structure of OCRL and INPP5B. E: TIRF microscopy of Cos-7 cells transfected with EGFP-clathrin light chain together with RFP-OCRL (left column) or RFP-INPP5B (right column). OCRL, but not INPP5B, colocalizes with a subset of clathrin spots in these single short exposure (200 msec) frames. F: Percentage of clathrin coated pits positive for INPP5B or for OCRL during their lifetime. G and H: Sequential TIRF microscopy images (8 sec intervals)(G) and time course of fluorescence intensity (H) for two clathrin-coated pits from cells co-transfected with RFP-clathrin and EGFP-OCRL. Note: In H, fluorescence is measured at a specific position so that both loss of fluorescence and lateral movement of the fluorescent spot away from such position result in a loss of signal intensity. I: Interaction of clathrin heavy chain with OCRL but not with INPP5B as revealed by western blotting of material affinity-purified by GST-OCRL or GST-INPP5B from a rat brain lysate (Bars = A, 10 μm; B, 2.5 μm; C, 5 μm; E, 5 μm; F, 1 μm).

A: Schematic drawing indicating direct and indirect interactions of OCRL described in this study or reported in the literature. The PDZ domain of GIPC binds the COOH-terminus of both the TrkA receptor and megalin. The PTB domain of APPL1 also binds the cytoplasmic domain of TrkA. A direct interaction of this PTB domain with megalin remains hypothetical. B: GST pulldowns of a rat brain lysate on GST or GST fusions of the COOH-terminal region of OCRL (top) or of the cytoplasmic domain of megalin (bottom). The western blots shown demonstrate presence of both APPL1 and GIPC in the materials bound both baits. C: Protein network involving OCRL and APPL1 and potentially relevant to the kidney and brain defects observed in Lowe syndrome. Loss of function mutations of proteins indicated in red lead to renal proximal tubule reabsorption defects either in human (OCRL) or in mouse (Megalin, GIPC and Dab2)(see text). D: Presence of APPL1 in both rat brain and kidney lysates (western blot). E: Immunofluorescence of cross-sectioned (cryostat sections) mouse kidney proximal tubules showing enrichment of APPL1 at the apical region of the epithelium, where it colocalizes with GIPC, myosin VI, and megalin, and also, partially with γ-adaptin. (Bar = 10 μm)

A: Ribbon diagram with the ASH domain (red) and the RhoGAP-like domain (blue). Residues of the clathrin box motif are in sticks. B: Stereo view of the C^α backbone traces. Trace color ramped from the NH2 terminus (red) to the COOH-terminus (blue). Every 20 residues are labeled and indicated with small filled circles. C: (Left) RhoGAP like domain of OCRL (blue) superimposed on the RhoGAP domain of P50RhoGAP (PDB ID: 1AM4). Bound Cdc42 is shown in gold. (Right) Biochemical interaction of the RhoGAP-like domain of OCRL and INPP5B with Rac and Cdc42. GST pulldowns from lysates of Cos-7 cells expressing tagged versions of Rac, Rho or Cdc42 with GST-fusions of the RhoGAP-like domain of OCRL or INPP5B. Bound proteins were analyzed by western-blotting. D: Alignment of the ASH domain of OCRL and INPP5B with other members of the MSP/VAP domain protein family. The E585 deletion mutation found in a patient is marked by a black triangle. Secondary structure elements are drawn above the alignment. Entrez database accession numbers are as follows: OCRL_Hs, GI: 57209431; Inpp5_Hs, GI: 59803021; OCRL_Dr, GI: 68374521; Inpp5_Dm, GI: 54642833; Inpp5_Ce, GI: 17505597; Inpp5_Dd, GI: 66828629; MSP_Ce, GI: 21730216; Vap-A_Rn, GI: 73535850. (Hs, Homo sapiens; Dr, Danio rerio; DM, Drosophila melanogaster; CE, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Rn, Rattus norvegicus).

The ASH-RhoGAP-like domains of OCRL were docked to the structure of the inositol 5-phosphastase domain from S. Pombe determined by Hurley and coworkers (Tsujishita et al., 2001). The NH2-terminal domain of OCRL, whose structure remains unknown, is not included. OCRL and INPP5B form an arc that is anchored to the membrane via the catalytic site of the inositol 5-phosphatase domain (shown here in a complex with the head group of a phosphoinositide) (PDB ID: 1I9Z) and the COOH-terminal region of the RhoGAP-like domain, which in INPP5B is farnesylated. Three mutations which abolish Rab5 binding are located within a small region (solid oval line), whose distance from the bilayer can accommodate a small GTPase such as Rab5. Rac or Cdc42 could also be accommodated next to the RhoGAP-like domain. The precise binding interface for APPL1 (dashed oval line) remains to be defined. The clathrin binding site protrudes from the structure.