ABSTRACT
The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.
Subject(s)
Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing , Carrier Proteins , HeLa Cells , Humans , Immunohistochemistry , Plasmids , Protein Binding , Two-Hybrid System TechniquesABSTRACT
Muscarinic acetylcholine receptors (mAChR) are involved in learning and memory but their molecular function in these processes is not fully understood. In this study, the signal transduction pathway coupling mAChR activation to induction of the activity-regulated cytoskeleton-associated gene (ARC) was examined. ARC was first identified as an effector immediate early gene induced by neuronal activity and ARC protein is thought to play a role in synaptic plasticity. In rats, intraperitoneal injection of pilocarpine, a potent agonist of mAChR, led to increased ARC expression in the brain. In human SH-SY5Y neuroblastoma cells mAChR stimulation with carbachol caused a rapid and robust induction of ARC expression. This effect was inhibited by atropine, a nonselective muscarinic receptor antagonist as well as by M1/M3 subtype-specific antagonists. Analysis of mAChR downstream effectors revealed that protein kinase C (PKC) and tyrosine kinases of the src family are key molecules in the signal cascade leading to ARC expression. Our data suggest, for the first time, that a correlation exists among mAChR-controlled signal cascades, the induction of the effector immediate early gene ARC and synaptic plasticity.
Subject(s)
Immediate-Early Proteins/metabolism , Nerve Tissue Proteins , Receptors, Muscarinic/physiology , Animals , Blotting, Northern/methods , Brain/drug effects , Brain/metabolism , Cell Line, Tumor , Cholinergic Agents/pharmacology , Cysteine-Rich Protein 61 , Cytoskeletal Proteins , Drug Interactions , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Immediate-Early Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Male , Muscarinic Agonists/pharmacology , Neuroblastoma , Pilocarpine/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Signal Transduction/drug effects , Time FactorsABSTRACT
Two isoforms of alpha-actinin 1 (ACTN1) known to be generated by tissue-specific alternative splicing of mutually exclusive exons have been described. Muscle cells express ACTN1 containing the smooth muscle exon (SM), while other (non-muscle) cells contain the non-muscle exon (NM). In this report, we describe the characterization of a novel ACTN1 isoform in adult rat brain in which both exons (NM + SM) are combined in the same transcript to give a brain-specific sequence domain (BS). Reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that expression of the BS exon was restricted to the brain. During development, weak expression of the BS exon was observed at early postnatal stages whereas in adult brain, it represented the predominant isoform of ACTN1. In situ hybridization analysis revealed that BS expression was highest in neurons of the hippocampus, cortex, and caudate putamen while the cerebellum and other subcortical structures showed only weak labeling.
Subject(s)
Actinin/metabolism , Alternative Splicing , Brain/embryology , Brain/metabolism , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cerebral Cortex/metabolism , DNA, Complementary/metabolism , Exons , Hippocampus/metabolism , In Situ Hybridization , Models, Genetic , Molecular Sequence Data , Muscle, Smooth/metabolism , Neurons/metabolism , Protein Isoforms , RNA/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
We examined the effect of insulin on the expression of the activity-regulated cytoskeleton-associated gene (ARC), an effector immediate early gene with a proposed role in memory formation. In human SH-SY5Y neuroblastoma cells, application of insulin leads to dramatic increase in ARC mRNA and protein levels. Inhibition experiments reveal, that p21(ras), mitogen-activated protein kinase/extracellular regulated kinase and tyrosine kinase (src) activity are required for the insulin-induced ARC expression in SH-SY5Y cells, whereas protein kinase C is not involved in the signal transduction pathway. Our data indicated for the first time a correlation of the insulin-controlled signal cascade and the induction of synaptic plasticity-associated immediate early genes.
