All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding

Abstract

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease.

Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1

“…Because of the strong level of evolutionary conservation between the ASH-RhoGAP-like modules of invertebrate and vertebrate OCRL (Fig. S1), including the conservation of those amino acids whose mutations are responsible for Lowe syndrome (29)(30)(31)(36)(37)(38), and this paper), it seemed reasonable to assume that this module originally had evolved to bind proteins other than APPL1. In scanning the literature, it became apparent that one candidate interactor of OCRL had been isolated previously in a genome-wide yeast twohybrid screen of Drosophila melanogaster proteins (39).…”

“…Some mutations produce absence of the protein or either truncations or functional disruption of its inositol 5′ phosphatase domain (http://research.nhgri.nih.gov/lowe/). However, other mutations are truncations or missense mutations in the Cterminal ASH-RhoGAP-like module, indicating that this region, which is important for several protein interactions, is critical for function (29,30) and that aberrant binding properties of missense mutations can compound the deficit caused by reduction in levels of protein expression commonly found in these mutations (17). Importantly, fibroblasts from Lowe syndrome patients display an increase in the lipid substrate PI(4,5)P 2 , regardless of the kind of mutation (31-33).…”

“…Importantly, fibroblasts from Lowe syndrome patients display an increase in the lipid substrate PI(4,5)P 2 , regardless of the kind of mutation (31-33). This finding supports the idea that not only inositol 5′ phosphatase activity, but also proper regulation and localization of this activity via protein-protein interactions, is required for OCRL to act on specific phosphoinositide pools.Recent work has identified the Rab5 effector APPL1 (Adaptor protein containing PH domain, PTB domain and Leucine Zipper motif 1) as a ligand of the ASH-RhoGAP-like domain (20,29). APPL1 resides on a subset of peripheral OCRL-positive endosomes (20, 34) that represent the earliest stations of endocytic traffic for a subset of endocytic vesicles (35).…”

“…Recent work has identified the Rab5 effector APPL1 (Adaptor protein containing PH domain, PTB domain and Leucine Zipper motif 1) as a ligand of the ASH-RhoGAP-like domain (20,29). APPL1 resides on a subset of peripheral OCRL-positive endosomes (20, 34) that represent the earliest stations of endocytic traffic for a subset of endocytic vesicles (35).…”

“…This subset includes vesicles derived from clathrin-coated pits that internalize growth factor receptors and macropinosomes that originate from the activation of growth factor receptors and/or their downstream signaling machinery (35). APPL1 binding was shown to be the only known OCRL interaction that is abolished by several mutations of the ASH-RhoGAP-like domain leading to Lowe syndrome (20,29), suggesting its key importance to OCRL function. However, APPL1 is present only in the vertebrate lineage, whereas the OCRL ASH-RhoGAP-like module is conserved throughout evolution, and the majority of amino acids required for APPL1 binding are preserved in the Drosophila ASH-RhoGAP-like domain.…”