Kai S. Erdmann

Identification and Characterization of the Human Orthologue of Yeast Pex14p

ABSTRACT Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makesHsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.

Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor

The Trk family of receptor tyrosine kinases and the p75 receptor (p75NTR) mediate the effects of neurotrophins on neuronal survival, differentiation and synaptic plasticity. The neurotrophin BDNF and its cognate receptor tyrosine kinase, TrkB.FL, are highly expressed in neurons of the central nervous system. At later stages in postnatal development the truncated TrkB splice variants (TrkB.T1, TrkB.T2) become abundant. However, the signalling and function of these truncated receptors remained largely elusive. We show that overexpression of TrkB.T1 in hippocampal neurons induces the formation of dendritic filopodia, which are known precursors of synaptic spines. The induction of filopodia by TrkB.T1 occurs independently of neurotrophin binding and of kinase activity of endogenous TrkB.FL. Coexpression of a p75NTR lacking an intracellular domain inhibits the TrkB.T1-induced effect in a dominant negative manner. Steric hindrance of extracellular p75NTR interactions with a specific antibody, or absence of p75NTR with an intact extracellular domain also inhibit this TrkB.T1-induced effect. We thus propose a novel signalling pathway initiated by neurotrophin-independent extracellular or intramembrane interaction of TrkB.T1 with the p75NTR receptor, which modulates dendritic growth via p75NTR signalling cascades.

The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain

Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein–protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (KD) of 8.1×10−9 M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as β-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

A structural basis for Lowe syndrome caused by mutations in the Rab‐binding domain of OCRL1

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X‐linked disease caused by mutations of the inositol‐5‐phosphatase OCRL1. OCRL1 is a membrane‐bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab‐binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α‐helical structure elements, the Rab‐binding interface of OCRL1 consists mainly of the IgG‐like β‐strand structure of the ASPM‐SPD‐2‐Hydin domain as well as one α‐helix. Our results give a deeper structural understanding of disease‐causing mutations of OCRL1 affecting Rab binding.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase‐basophil like (PTP‐BL) is a large non‐transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP‐BL with the protein kinase C‐related kinase 2 (PRK2), a serine/threonine kinase regulated by the G‐protein rho. This interaction is mediated by the PSD‐95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP‐BL and the extreme C‐terminus of PRK2 as shown by yeast two‐hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C‐terminal cysteine of PRK2 is indispensable for the interaction with PTP‐BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP‐BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP‐BL in the regulation of the actin cytoskeleton.

RNAi screening identifies mediators of NOD2 signaling: Implications for spatial specificity of MDP recognition

The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95

Semaphorins are a family of secreted and membrane‐associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post‐synaptic density protein SAP90/PSD‐95. Using the yeast two‐hybrid system and coprecipitation assays we were able to show an interaction between the extreme C‐terminus of Sema4F and the PDZ domains of SAP90/PSD‐95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD‐95 in COS cells leads to translocation of SAP90/PSD‐95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C‐terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post‐synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD‐95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

Crystal structure of the Rab binding domain of OCRL1 in complex with Rab8 and functional implications of the OCRL1/Rab8 module for Lowe syndrome

Mutations of the inositol-5-phosphatase OCRL1 cause Lowe syndrome. Lowe syndrome is an inherited disease characterized by renal dysfunction and impaired development of the eye and the nervous system. OCRL1 is a Rab effector protein that can bind to a large number of different Rab proteins. We have recently determined the X-ray structure of the Rab-binding domain of OCRL1 in complex with Rab8. Furthermore, we have characterized point mutations that abolish binding to Rab proteins and cause Lowe syndrome. Here we shortly review our recent biophysical and structural work and discuss possible functional implications of our finding that Rab8 binds with the highest affinity to OCRL1 among the Rab proteins tested. This could direct further work on OCRL1 leading to a better understanding of the complex disease mechanism of Lowe syndrome.

PDZ-domain-directed basolateral targeting of the peripheral membrane protein FRMPD2 in epithelial cells

Multi-PDZ (PSD-95/Discs large/Zonula-occludens-1) domain proteins play a crucial role in the establishment and maintenance of cell polarization. The novel multi-PDZ domain protein FRMPD2 is a potential scaffolding protein consisting of an N-terminal KIND domain, a FERM domain and three PDZ domains. Here we show that FRMPD2 is localized in a polarized fashion in epithelial cells at the basolateral membrane and partially colocalizes with the tight-junction marker protein Zonula-occludens-1. Downregulation of FRMPD2 protein in Caco-2 cells is associated with an impairment of tight junction formation. We find that the FERM domain of FRMPD2 binds phosphatidylinositols and is sufficient for membrane localization. Moreover, we demonstrate that recruitment of FRMPD2 to cell-cell junctions is strictly E-cadherin-dependent, which is in line with our identification of catenin family proteins as binding partners for FRMPD2. We demonstrate that the FERM domain and binding of the PDZ2 domain to the armadillo protein p0071 are required for basolateral restriction of FRMPD2. Moreover, the PDZ2 domain of FRMPD2 is sufficient to partially redirect an apically localized protein to the basolateral membrane. Our results provide novel insights into the molecular function of FRMPD2 and into the targeting mechanism of peripheral membrane proteins in polarized epithelial cells.