The NH2-terminal region of OCRL binds to clathrin. (A) Schematic diagram showing domain structure of INPP5B and OCRL and the GST-fusion constructs used for pull-downs. Sequence similarity between INPP5B and OCRL is listed for each pair of homologous domains. (B) GST and GST-OCRL NH2-terminal regions were incubated with rat brain lysate, and bound proteins were analysed by SDS–PAGE, followed by Coomassie blue staining. Protein bands were identified by mass spectrometry and confirmed by western blotting. CHC, clathrin heavy chain; AP-2 followed by a Greek symbol, subunits of the clathrin adaptor AP-2. (C) GST pull-downs as in (B) using GST-OCRL¹⁻¹⁴¹ wild-type (WT) or with single point mutations in the sequence ⁷²LLIDIA⁷⁷. Binding to clathrin (CHC) is abolished by mutations of hydrophobic residues of the motif ⁷³LIDI⁷⁶, and partially impaired by mutation of the leucine upstream of the motif. (D) SPR analysis of the binding of various concentrations (0–400 nM) of the NH2-terminal β-propeller domain of clathrin heavy chain to immobilized GST-OCRL¹⁻¹¹⁹. The calculated K_D value is indicated. (E) Binding of clathrin's propeller domain (400 nM) to GST-OCRL¹⁻¹¹⁹ after preincubation with the module itself (GST-OCRL¹⁻¹¹⁹, 4 μM), with the clathrin-box peptide contained in this module (ETLLIDIASNS) (8 μM) or with a peptide from the clathrin adaptor AP-2 comprising a classical clathrin-box motif (GDLLNLDLGP) (8 μM).

NMR structure of the NH2-terminal PH domain of OCRL. (A) Superimposed C^α backbone traces of the 20 lowest-energy calculated structures. Traces are rainbow coloured with the NH2-terminus in red and the COOH-terminus in blue. Numbers and corresponding small filled circles indicate residues multiple of 20. (B) Ribbon diagram of a representative structure from (A) shown in the same orientation. Residues that are involved in clathrin binding are shown in sticks and labelled. (C) Ribbon diagram of the structure of the PH domain of PLCδ (PDB ID 1mai). (D) Sequence alignment of the NH2-terminal PH domains of OCRL orthologs. Secondary elements are drawn under the alignment. The clathrin-biding motif is marked by red triangles. This motif, as well as the preceding leucine that partially contributes to binding (see Figure 1C) is completely conserved among all OCRL orthologs. Entrez database accession numbers are as follow: OCRL_PP, GI: 55729733; OCRL_CF, GI: 74008389; OCRL_BT, GI: 156121029; OCRL_MM, GI: 45768389; OCRL_EC, GI: 149745642; OCRL_GG, GI: 118089331; OCRL_XT, GI: 115529017; OCRL_DR, GI: 118150544; OCRL_HS, GI: 13325072. PP, Pongo pygmaeus; CF, Canis familiaris; BT, Bos Taurus; MM, Mus musculus; EC, Equus caballus; GG, Gallus gallus; XT, Xenopus tropicalis; DR, Danio rerio; HS, Homo sapiens.

NMR structure of the NH2-terminal PH domain of INPP5B. (A) C^α backbone traces of the 20 lowest-energy structures of the NH2-terminal PH domain of INPP5B in superimposition. Traces are rainbow coloured with the NH2-terminus in red and the COOH-terminus in blue. Numbers and corresponding small filled circles indicate residues multiple of 20. (B) Ribbon diagram of a representative structure from (A). (C) Structure comparison of the PH domains from OCRL (in green and red) and from INPP5B (in blue and pink). (D) Sequence alignment of the NH2-terminal PH domains of INPP5B orthologs in several species. The human OCRL PH domain sequence is alignment on the bottom line based on 3D structure. Secondary elements are drawn under the alignment. Entrez database accession numbers are as follow: INPP5B_AP, GI: 193671757; INPP5B_AM, GI: 110761623; INPP5B_HS, GI: 55666459; INPP5B_ BT, GI: 194665894; INPP5B_AA, GI: 157134484; INPP5B_ DM, 24639220; INPP5B_ SP, GI: 115947205; INPP5B_MM, GI: 30231213; OCRL_HS, GI: 13325072. AP, Acyrthosiphon pisum; AM, Apis mellifera; HS, Homo sapiens, BT, Bos Taurus; AA, Aedes aegypti; DM, Drosophila melanogaster; SP, Strongylocentrotus purpuratus; MM, Mus musculus.

The PH domains of OCRL and INPP5B lack the basic PI(4,5)P₂ binding pocket present in the PH domain of PLCδ. Positive and negative electrostatic surface potentials are indicated in blue and red, respectively. The PH domain of PLCδ with bound IP3 (PDB ID: 1mai) is from Ferguson et al (1995).

Overexpression of EGFP-OCRL¹⁻¹¹⁹ impairs endocytosis of transferrin. (A) Hela cells were transfected with expression vectors for the indicated GFP-fusion proteins. At 48 h after transfection, cells were incubated with transferrin-Alexa594 for 10 min. After washing and acid stripping, cells were fixed and inspected by fluorescence microscopy. Arrows indicate transfected cells demonstrating reduced transferrin uptake after EGFP-OCRL¹⁻¹¹⁹ transfection and unchanged transferrin uptake after EGFP-OCRL¹⁻¹¹⁹ I74N transfection. Scale bar is 10 μm. (B) Quantification of transferrin uptake. Hela cells were transfected with expression vectors for EGFP-OCRL¹⁻¹¹⁹ (filled squares) or EGFP-OCRL¹⁻¹¹⁹ I74N (filled triangles). After 48 h, uptake of biotinylated transferrin was measured at the indicated time points using an ELISA assay. Values are the mean±s.e. (n=3).

Recruitment of OCRL to clathrin-coated endocytic pits as assessed by TIRF microscopy. (A) Schematic representation of the domain architecture of OCRL/INPP5B and of the mutant constructs used in TIRF microscopy experiments. Red strikethrough indicates disruption of the corresponding clathrin coat-binding site by mutagenesis (see Materials and methods). (B) Percentage of clathrin-coated pits (LCa-mRFP) that clearly become positive for GFP construct indicated during their final stages of maturation. (C) Representative TIRF microscopy images of wild-type and mutant (I74N) OCRL. The I74N mutation dramatically disrupts OCRL localization resulting in a loss of peripheral dots (clathrin-coated pits) and a higher cytosolic pool. Many of the dots still visible (inset) are highly mobile, most likely representing Rab5-bound OCRL on early endosomes. (D) Kymographs of cells coexpressing the indicated GFP-OCRL/INPP5B proteins (green) and LCa-mRFP (red). Individual clathrin-coated pits appear as horizontal red traces. A green end of the trace indicates the recruitment of the GFP-fusion protein before pit disappearance.

Model of the full-length OCRL at the membrane interface. The inositol 5-phosphatase domain together with the ASH and RhoGAP-like domains are docked at the membrane interface as proposed earlier (Erdmann et al, 2007). The NH2-terminal PH domain of OCRL is connected to the 5-phosphatase domain by a long flexible linker region. An FxDxF motif within this region mediates binding to the endocytic clathrin adaptor AP-2. The β-propeller NH2-terminal region of clathrin binds directly to OCRL through two clathrin boxes in the NH2-terminal and C-terminal region of OCRL, respectively, and indirectly through AP-2. Binding site for Rab5 and APPL1 in OCRL are also indicated.