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Differential localization of G protein ?? subunits.

ABSTRACT: G protein ?? subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both G? and G? isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of G? and G? isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular G? and G? subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most G? and G? isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.

SUBMITTER: Betke KM

PROVIDER: S-EPMC4004276 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Differential localization of G protein βγ subunits.

Betke Katherine M KM Rose Kristie L KL Friedman David B DB Baucum Anthony J AJ Hyde Karren K Schey Kevin L KL Hamm Heidi E HE

Biochemistry 20140401 14

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics appr ...[more]

PMID: 24568373

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Project description:BACKGROUND:ATP-sensitive K+ (KATP) channels were originally found in cardiac myocytes by Noma in 1983. KATP channels were formed by potassium ion-passing pore-forming subunits (Kir6.1, Kir6.2) and regulatory subunits SUR1, SU2A and SUR2B. A number of cells and tissues have been revealed to contain these channels including hepatocytes, but detailed localization of these subunits in different types of liver cells was still uncertain. AIM:To investigate the expression of KATP channel subunits in rat liver and their localization in different cells of the liver. METHODS:Rabbit anti-rat SUR1 peptide antibody was raised and purified by antigen immunoaffinity column chromatography. Four of Sprague-Dawley rats were used for liver protein extraction for immunoblot analysis, seven of them were used for immunohistochemistry both for the ABC method and immunofluorescence staining. Four of Wistar rats were used for the isolation of hepatic stellate cells (HSCs) and Kupffer cells for both primary culture and immunocytochemistry. RESULTS:Immunoblot analysis showed that the five kinds of KATP channel subunits, i.e. Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B, were detected in liver. Immunohistochemical staining showed that Kir6.1 and Kir6.2 were weakly to moderately expressed in parenchymal cells and sinusoidal lining cells, while SUR1, SUR2A, and SUR2B were mainly localized to sinusoidal lining cells, such as HSCs, Kupffer cells, and sinusoidal endothelial cells. Immunoreactivity for SUR2A and SUR2B was expressed in the hepatocyte membrane. Double immunofluorescence staining further showed that the pore-forming subunits Kir6.1 and/or Kir6.2 colocalized with GFAP in rat liver sections and primary cultured HSCs. These KATP channel subunits also colocalized with CD68 in liver sections and primary cultured Kupffer cells. The SUR subunits colocalized with GFAP in liver sections and colocalized with CD68 both in liver sections and primary cultured Kupffer cells. In addition, five KATP channel subunits colocalized with SE-1 in sinusoidal endothelial cells. CONCLUSION:Observations from the present study indicated that KATP channel subunits expressed in rat liver and the diversity of KATP channel subunit composition might form different types of KATP channels. This is applicable to hepatocytes, HSCs, various types of Kupffer cells and sinusoidal endothelial cells.

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Differential localization of G protein ?? subunits.

Publications

Differential localization of G protein βγ subunits.

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