Lisa A. Elferink

The syntaxin family of vesicular transport receptors

Syntaxins A and B are nervous system-specific proteins implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. A family of syntaxin-related proteins from rat has been identified that shares 23%-84% amino acid identity. Each of the six syntaxins terminate with a carboxy-terminal hydrophobic domain that anchors the protein on the cytoplasmic surface of cellular membranes. The syntaxins display a broad tissue distribution and, when expressed in COS cells, are targeted to different subcellular compartments. Microinjection studies suggest that the nervous system-specific syntaxin 1A is important for calcium-regulated secretion from neuro-endocrine PC12 cells. These results indicate that the syntaxins are a family of receptors for intracellular transport vesicles and that each target membrane may be identified by a specific member of the syntaxin family.

A role for synaptotagmin (p65) in regulated exocytosis

Proteins that are specifically localized to synaptic vesicles in the nervous system have been proposed to mediate aspects of synaptic transmission. Antibodies raised against the cytoplasmic domains of five of these proteins, vamp, rab3A, synaptophysin, synaptotagmin, and SV2, were used to investigate their function. Microinjection of monoclonal and polyclonal antibodies raised against synaptotagmin (p65), but not the other vesicle proteins, decreases K+/Ca(2+)-mediated dopamine beta-hydroxylase surface staining, a measure of regulated secretion in PC12 cells. Microinjection of a soluble fragment of synaptotagmin encompassing one of the domains homologous to the C2 regulatory region of protein kinase C, but lacking the membrane anchor, also inhibits evoked dopamine beta-hydroxylase surface staining. These results provide support for the hypothesis that synaptotagmin, a Ca(2+)- and phospholipid-binding protein, is important for regulated exocytosis in neurons.

Tyrosine hydroxylase is inactivated by catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to Parkinson's disease.

Abstract Quinone derivatives of DOPA, dopamine, and N‐acetyldopamine inactivate tyrosine hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl‐catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox‐cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton‐like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox‐active species is highly relevant for neurodegenerative conditions such as Parkinson’s disease.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination

Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCα along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCα with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans.

Rab15 mediates an early endocytic event in Chinese hamster ovary cells.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.

Association of three small GTP-binding proteins with cholinergic synaptic vesicles

Several small (low molecular weight) GTP‐binding proteins are associated with cholinergic synaptic vesicles derived from the electric organ of electric ray. Using GTP overlay techniques and direct micro sequencing we analyzed the association of small GTP‐binding proteins with synaptic vesicles. Both experimental procedures revealed the specific occurrence of multiple small GTP‐binding proteins with this organelle. Moreover, direct amino acid sequence analysis assigned at least three different small GTP‐binding proteins, ora3, o‐ral and o‐rab3, to the vesicular compartment. Furthermore, the data reflect the relative abundance of these three proteins on the vesicle membrane, thereby demonstrating the predominant occurrence of o‐rab3, the only exclusively synaptic vesicle specific small GTP‐binding protein.

Synthesis, Degradation, and Subcellular Localization of Synaptotagmin IV, a Neuronal Immediate Early Gene Product

Abstract: Synaptotagmin IV (Syt IV) is an immediate early gene induced by depolarization in rat PC12 cells and in rat hippocampus. We prepared an antiserum to Syt IV protein. The 46‐kDa Syt IV protein is nearly undetectable by western blotting in unstimulated PC12 cells. After depolarization, Syt IV increases rapidly, peaks at 4 h, and decays to near baseline levels by 12 h. Forskolin stimulation also leads to rapid Syt IV protein accumulation. The rate of Syt IV protein synthesis, determined by labeling with radioactive amino acids and immunoprecipitation, is low in unstimulated PC12 cells, but increases over the first 3 h after forskolin stimulation and remains elevated for several hours. Syt IV protein is relatively labile; metabolically labeled Syt IV has a half‐life of ∼2 h in PC12 cells. Sucrose density gradient fractionation and vesicle immunoisolation experiments suggest that Syt IV protein is present in both synaptic‐like microvesicles and secretory granules. Vesicles immunoisolated from forskolin‐treated PC12 cells with anti‐Syt I antibody contain radioactively labeled Syt IV, demonstrating that Syt I and Syt IV colocalize in common vesicles. These results suggest that Syt IV protein, after its stimulation‐induced synthesis, is rapidly transported to secretory vesicles where it may transiently modulate the exocytotic machinery.

The Listeria Protein Internalin B Mimics Hepatocyte Growth Factor‐Induced Receptor Trafficking

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein of Listeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF‐induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin‐dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol‐chelating drug Nystatin. Thus, HGFR internalization is principally clathrin‐mediated and is not regulated by clathrin‐ independent pathways. Phosphatidylinositol 3‐kinase signaling and HGF‐regulated tyrosine kinase substrate were not required for ligand‐triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling.

Specific Grb2-mediated interactions regulate clathrin-dependent endocytosis of the cMet-tyrosine kinase

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.