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1.
J Biol Chem ; 288(16): 11144-54, 2013 Apr 19.
Article in English | MEDLINE | ID: mdl-23471969

ABSTRACT

Facilitation of nerve growth factor (NGF) signaling by the p75 neurotrophin receptor (p75(NTR)) is critical for neuronal survival and differentiation. However, the interaction between p75(NTR) and TrkA receptors required for this activity is not understood. Here, we report that a specific 29-amino acid peptide derived from the intracellular domain fragment of p75(NTR) interacts with and potentiates binding of NGF to TrkA-expressing cells, leading to increased neurite outgrowth in sympathetic neurons as a result of enhanced Erk1/2 and Akt signaling. An endogenous intracellular domain fragment of p75(NTR) (p75(ICD)) containing these 29 amino acids is produced by regulated proteolysis of the full-length receptor. We demonstrate that generation of this fragment is a requirement for p75(NTR) to facilitate TrkA signaling in neurons and propose that the juxtamembrane region of p75(ICD) acts to cause a conformational change within the extracellular domain of TrkA. This finding provides new insight into the mechanism by which p75(NTR) and TrkA interact to enhance neurotrophic signaling.


Subject(s)
MAP Kinase Signaling System/physiology , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptor, trkA/metabolism , Receptors, Nerve Growth Factor/metabolism , Animals , HEK293 Cells , Humans , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Neurons/cytology , Protein Structure, Tertiary , Proteolysis , Receptor, trkA/genetics , Receptors, Nerve Growth Factor/genetics
2.
J Biol Chem ; 287(52): 43810-24, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23105112

ABSTRACT

Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75(NTR), best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75(NTR) ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75(NTR) are γ-secretase substrates. However, dimerization is not a requirement for p75(NTR) cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75(NTR) interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75(NTR) transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75(NTR). Nonetheless, heteromer interactions of p75(NTR) with TrkA increase full-length p75(NTR) homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75(NTR) transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Nerve Tissue Proteins/metabolism , Protein Multimerization/physiology , Proteolysis , Receptors, Growth Factor/metabolism , Receptors, Nerve Growth Factor/metabolism , Amino Acid Motifs , Amyloid Precursor Protein Secretases/genetics , Animals , Cell Death/physiology , Cysteine , Enzyme Activation , HEK293 Cells , Humans , Nerve Growth Factor/genetics , Nerve Growth Factor/metabolism , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/genetics , PC12 Cells , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Structure, Tertiary , Rats , Receptor, trkA/genetics , Receptor, trkA/metabolism , Receptors, Growth Factor/genetics , Receptors, Nerve Growth Factor/genetics
3.
J Biol Chem ; 287(52): 43798-809, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23105113

ABSTRACT

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75(NTR)). To identify the interaction site between sortilin and p75(NTR), we analyzed binding between chimeric receptor constructs and truncated p75(NTR) variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75(NTR) relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75(NTR). Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75(NTR)-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75(NTR) binding, it does regulate the rates of p75(NTR) cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Apoptosis/physiology , Nerve Tissue Proteins/metabolism , Proteolysis , Receptors, Nerve Growth Factor/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Animals , Cell Line, Tumor , HEK293 Cells , Humans , Nerve Tissue Proteins/genetics , Peptide Mapping , Protein Structure, Tertiary , Rats , Receptors, Nerve Growth Factor/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Surface Plasmon Resonance
4.
Biometals ; 23(4): 681-94, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20372979

ABSTRACT

Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.


Subject(s)
Adenosine Triphosphatases/metabolism , Cation Transport Proteins/metabolism , Copper/metabolism , Adenosine Triphosphatases/genetics , Animals , Biological Transport , Cation Transport Proteins/genetics , Cells, Cultured , Copper-Transporting ATPases , Dogs , Drosophila melanogaster/enzymology , Humans , Menkes Kinky Hair Syndrome/genetics , Menkes Kinky Hair Syndrome/metabolism , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism
5.
Int J Biochem Cell Biol ; 41(12): 2403-12, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19576997

ABSTRACT

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.


Subject(s)
Adenosine Triphosphatases/metabolism , Cation Transport Proteins/metabolism , Copper/metabolism , Kidney/metabolism , Ovary/metabolism , Animals , Copper-Transporting ATPases , Cricetinae , Dogs , Female , Humans , Kidney/pathology , Mice , Mutagenesis, Site-Directed , Ovary/pathology , Phosphorylation , Protein Transport , Signal Transduction
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