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P S McPherson

Publications and source records attributed to P S McPherson.

At least 19 recordsLinked to original sources
PubMed2004

Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics.

Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain. Accounting for identified fragmented peptides allows for a quantitative assessment of protein complexes associated with CCVs, and the identification of many of the known components of post-fusion synaptic vesicles demonstrates that a main function for brain CCVs is to recycle synaptic vesicles. In addition, we have identified a number of novel proteins that participate in CCV formation and function at the trans-Golgi network and the plasma membrane. Characterization of two of these proteins, NECAP1 and NECAP2, has led to the identification of a new consensus motif that mediates protein interactions with the clathrin adaptor protein 2. These studies highlight the ability of proteomics to reveal new insights into the mechanisms and functional roles of subcellular compartments.

Adaptor Protein Complex 2↗
PubMed2001

HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2.

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

Adaptor Protein Complex 2↗
PubMed2001

Parathyroid hormone-related peptide stimulates osteogenic cell proliferation through protein kinase C activation of the Ras/mitogen-activated protein kinase signaling pathway.

We investigated the mechanisms of parathyroid hormone-related peptide (PTHrP)-mediated effects on osteogenic cells in primary rat bone marrow cell (BMC) cultures. We first demonstrated by reverse transcriptase-polymerase chain reaction and immunocytochemistry that BMCs express the type I parathyroid hormone/PTHrP receptor. Treatment with PTHrP increased osteogenic cell proliferation as determined by [(3)H]thymidine and bromodeoxyuridine incorporation and augmented osteogenic colonies. Immunocytochemistry and Western blotting revealed no direct effect on expression of the osteoblast markers, type I collagen, bone sialoprotein, and osteocalcin, indicating that PTHrP did not directly stimulate differentiation in this system. PTHrP increased mitogen-activated protein kinase (MAPK) activity in BMC and MAPK activity, and PTHrP-induced osteogenic cell proliferation could be blocked by the MEK inhibitor PD-098059. PTHrP also increased Ras activity in BMC. Although wortmannin and H8, inhibitors of phosphoinositol 3-kinase and protein kinase A, respectively, did not block PTHrP-stimulated Ras or MAPK activity, chelerythrin chloride, a known protein kinase C inhibitor, did block these PTHrP actions as well as PTHrP-induced osteogenic cell proliferation. These results demonstrate that PTHrP stimulates osteogenic cell proliferation in rat marrow mesenchymal progenitor cells through protein kinase C-dependent activation of the Ras and MAPK signaling pathway.

Alkaline Phosphatase↗
PubMed2001

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase.

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of c-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The Ras/Rac guanine nucleotide exchange factor mammalian Son-of-sevenless interacts with PACSIN 1/syndapin I, a regulator of endocytosis and the actin cytoskeleton.

Mammalian Son-of-sevenless (mSos) functions as a guanine nucleotide exchange factor for Ras and Rac, thus regulating signaling to mitogen-activated protein kinases and actin dynamics. In the current study, we have identified a new mSos-binding protein of 50 kDa (p50) that interacts with the mSos1 proline-rich domain. Mass spectrometry analysis and immunodepletion studies reveal p50 as PACSIN 1/syndapin I, a Src homology 3 domain-containing protein functioning in endocytosis and regulation of actin dynamics. In addition to PACSIN 1, which is neuron-specific, mSos also interacts with PACSIN 2, which is expressed in neuronal and nonneuronal tissues. PACSIN 2 shows enhanced binding to the mSos proline-rich domain in pull-down assays from brain extracts as compared with lung extracts, suggesting a tissue-specific regulation of the interaction. Proline to leucine mutations within the Src homology 3 domains of PACSIN 1 and 2 abolish their binding to mSos, demonstrating the specificity of the interactions. In situ, PACSIN 1 and mSos1 are co-expressed in growth cones and actin-rich filopodia in hippocampal and dorsal root ganglion neurons, and the two proteins co-immunoprecipitate from brain extracts. Moreover, epidermal growth factor treatment of COS-7 cells causes co-localization of PACSIN 1 and mSos1 in actin-rich membrane ruffles, and their interaction is regulated through epidermal growth factor-stimulated mSos1 phosphorylation. These data suggest that PACSINs may function with mSos1 in regulation of actin dynamics.

Actins↗
PubMed2001

Signaling on the endocytic pathway.

Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

Animals↗
PubMed2001

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

Actins↗
PubMed2000

Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis.

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 degrees C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.

Adaptor Proteins, Vesicular Transport↗
PubMed2000

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain.

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The N terminus of amphiphysin II mediates dimerization and plasma membrane targeting.

Amphiphysin I and II are nerve terminal-enriched proteins containing SH3 domains that interact with dynamin and synaptojanin. The amphiphysins may function in synaptic vesicle endocytosis by targeting synaptojanin and dynamin to emerging endocytic buds through SH3 domain-independent interactions with clathrin and AP2. We have recently identified and cloned several amphiphysin II splice variants that differentially incorporate clathrin-binding domains. To determine whether these domains function in membrane targeting, we used immunofluorescence to examine the potential localization of amphiphysin II variants to clathrin-coated pits on plasma membranes purified from transfected COS-7 cells. Full-length amphiphysin II targets to the plasma membrane where it partially co-localizes with clathrin. However, splice variants and deletion constructs lacking clathrin-binding domains still target to the plasma membrane, and removal of clathrin from the membrane does not affect amphiphysin II distribution. Surprisingly, plasma membrane targeting was dependent on the presence of a 31-amino acid alternatively spliced sequence at the N terminus of amphiphysin II, a result confirmed using subcellular fractionation. In binding assays, the 31-amino acid sequence was also found to facilitate amphiphysin dimerization mediated through the N terminus. Taken together, these data support a role for the N terminus of amphiphysin II in membrane targeting during endocytosis.

Alternative Splicing↗
PubMed1999

Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

Adaptor Protein Complex 2↗
PubMed1999

Regulatory role of SH3 domain-mediated protein-protein interactions in synaptic vesicle endocytosis.

Src homology (SH) 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein-protein interactions in this process.

Amino Acid Sequence↗
PubMed1999

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation.

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.

3T3 Cells↗
PubMed1998

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Adaptor Proteins, Signal Transducing↗
PubMed1998

A neuronal ryanodine receptor mediates light-induced phase delays of the circadian clock.

Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.

Animals↗
PubMed1998

EH domain-dependent interactions between Eps15 and clathrin-coated vesicle protein p95.

The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.

Amino Acid Sequence↗
PubMed1998

Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors.

Recent evidence suggests that extracellular ATP plays a neurotransmitter role in the central nervous system. Its fast ionotropic effects are exerted through a family of P2X ATP-gated channels expressed in brain and spinal cord. To determine the physiological significance of central ATP receptors, we have investigated the localization of a major neuronal P2X receptor at the cellular and subcellular levels using affinity-purified antibodies directed against the C-terminal domain of P2X4 subunit. Subunit-specific anti-P2X4 antibodies detected a single band of 57,000 +/- 3000 mol. wt in transfected HEK-293 cells and in homogenates from adult rat brain. The strongest expression of central P2X receptors was observed in the olfactory bulb, lateral septum, cerebellum and spinal cord. P2X4 immunoreactivity was also evident in widespread areas including the cerebral cortex, hippocampus, thalamus and brainstem. In all regions examined, P2X receptors were associated with perikarya and dendrites where they were concentrated at the level of afferent synaptic junctions, confirming a direct involvement of postsynaptic ATP-gated channels in fast excitatory purinergic transmission. Moreover, P2X4-containing purinoceptors were localized in axon terminals in the olfactory bulb and in the substantia gelatinosa of nucleus caudalis of the medulla and dorsal horn of the spinal cord, demonstrating an important selective presynaptic role of ATP in the modulation of neurotransmitter release in central sensory systems.

Adenosine Triphosphate↗