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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

ABSTRACT: Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

SUBMITTER: Cremona ML

PROVIDER: S-EPMC3066276 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Cremona M Laura ML Matthies Heinrich J G HJ Pau Kelvin K Bowton Erica E Speed Nicole N Lute Brandon J BJ Anderson Monique M Sen Namita N Robertson Sabrina D SD Vaughan Roxanne A RA Rothman James E JE Galli Aurelio A Javitch Jonathan A JA Yamamoto Ai A

Nature neuroscience 20110313 4

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter ( ...[more]

PMID: 21399631

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Project description:IntroductionDuring wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKC?/MLC-2). PKC? is activated by growth factor-driven PLC?1 hydrolysis of phosphoinositide bisphosphate (PIP2) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKC? n-terminus mediates PKC? localization to the membrane in relative proximity to PLC?1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKC? mediates the transcellular contractility of fibroblasts.MethodsTo determine PKC? activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKC? constructs were generated; PKC?-CaaX with farnesylation moiety targeting PKC? to the membrane and PKC?-SaaX a non-targeting control.ResultsIncreased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKC? (PKC?-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKC? (PKC?-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKC?.ConclusionFrom these results membrane tethering of PKC? leads to increased force exertion on ECM. Furthermore, our data show PLC?1 regulation of PKC?, at least in part, drives transcellular contractility in fibroblasts.

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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Publications

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

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