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Identification of KRAP-expressing cells and the functional relevance of KRAP to the subcellular localization of IP3R in the stomach and kidney.

Fujimoto T, Shirasawa S - Int. J. Mol. Med. (2012)

Bottom Line: We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas.Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs.These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

ABSTRACT
KRAS-induced actin-interacting protein (KRAP), originally identified as one of the deregulated genes expressed in colorectal cancer, participates under physiological conditions in the regulation of systemic energy homeostasis and of the exocrine system. We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas. However, the expression of KRAP and its precise function in other tissues remain elusive. In this study, we aimed to identify the KRAP-expressing cells in mouse stomach and kidneys and to examine the relevance of KRAP expression in the regulation of IP(3)R localization in these tissues. In the stomach, double immunohistochemical staining for KRAP and IP(3)R demonstrated that KRAP was expressed along with the apical regions in the mucous cells and the chief cells, and IP(3)R3 was dominantly co-localized with KRAP in these cells. Furthermore, IP(3)R2 was also co-localized with IP(3)R3 in the chief cells. It is of note that the proper localization of IP(3)R3 and IP(3)R2 in the chief cells and of IP(3)R3 in the mucous cells were significantly abrogated in KRAP-deficient mice. In the kidneys, KRAP was expressed in both the apical and the basal regions of the proximal tubular cells. Intriguingly, KRAP deficiency abrogated the localization of IP(3)R1 in the proximal tubular cells. Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs. These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

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Colocalization of KRAP with IP3Rs in the chief cells and the mucous cells ofthe mouse stomach. (A) Fluorescent confocal images of the base region of gastric glandsfor KRAP (red), IP3R3 (green), and the merged photo. Arrows indicate theapical membranes of the chief cells. (B) Fluorescent confocal images of the pit regionof gastric glands for KRAP (red), IP3R3 (green), and the merged photo. Arrowsindicate the apical membranes of the mucous cells. (C) Fluorescent confocal images ofthe base region of gastric glands for IP3R2 (red), IP3R3 (green),and the merged photo. Asterisks and arrow indicate the parietal cells and the apicalmembranes of the chief cells, respectively. (D) Fluorescent confocal images of the pitregion of gastric glands for IP3R2 (red), IP3R3 (green), and themerged photo. Arrows indicate the apical membranes of the mucous cells. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.
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f2-ijmm-30-06-1287: Colocalization of KRAP with IP3Rs in the chief cells and the mucous cells ofthe mouse stomach. (A) Fluorescent confocal images of the base region of gastric glandsfor KRAP (red), IP3R3 (green), and the merged photo. Arrows indicate theapical membranes of the chief cells. (B) Fluorescent confocal images of the pit regionof gastric glands for KRAP (red), IP3R3 (green), and the merged photo. Arrowsindicate the apical membranes of the mucous cells. (C) Fluorescent confocal images ofthe base region of gastric glands for IP3R2 (red), IP3R3 (green),and the merged photo. Asterisks and arrow indicate the parietal cells and the apicalmembranes of the chief cells, respectively. (D) Fluorescent confocal images of the pitregion of gastric glands for IP3R2 (red), IP3R3 (green), and themerged photo. Arrows indicate the apical membranes of the mucous cells. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.

Mentions: Since we previously reported that KRAP associates with particular subtypes ofIP3R in the liver and the pancreas (20), we examined whether KRAP in the stomach is also co-localizedwith IP3R. Double-immunostaining of the stomach for KRAP and IP3R3revealed that KRAP was co-localized with IP3R3 in the apical regions of boththe chief cells (Fig. 2A, arrows) andthe mucous cells (Fig. 2B, arrows).Of note, IP3R2 co-existed with IP3R3 in the chief cells (Fig. 2C, arrow) but not in the parietalcells (Fig. 2C, asterisks).Furthermore, IP3R2 was not detected in the mucous cells (Fig. 2D, arrows). These results indicatedthat KRAP was co-localized with IP3R2 and IP3R3 in the chief cellsand with IP3R3 in the mucous cells.


Identification of KRAP-expressing cells and the functional relevance of KRAP to the subcellular localization of IP3R in the stomach and kidney.

Fujimoto T, Shirasawa S - Int. J. Mol. Med. (2012)

Colocalization of KRAP with IP3Rs in the chief cells and the mucous cells ofthe mouse stomach. (A) Fluorescent confocal images of the base region of gastric glandsfor KRAP (red), IP3R3 (green), and the merged photo. Arrows indicate theapical membranes of the chief cells. (B) Fluorescent confocal images of the pit regionof gastric glands for KRAP (red), IP3R3 (green), and the merged photo. Arrowsindicate the apical membranes of the mucous cells. (C) Fluorescent confocal images ofthe base region of gastric glands for IP3R2 (red), IP3R3 (green),and the merged photo. Asterisks and arrow indicate the parietal cells and the apicalmembranes of the chief cells, respectively. (D) Fluorescent confocal images of the pitregion of gastric glands for IP3R2 (red), IP3R3 (green), and themerged photo. Arrows indicate the apical membranes of the mucous cells. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042864&req=5

f2-ijmm-30-06-1287: Colocalization of KRAP with IP3Rs in the chief cells and the mucous cells ofthe mouse stomach. (A) Fluorescent confocal images of the base region of gastric glandsfor KRAP (red), IP3R3 (green), and the merged photo. Arrows indicate theapical membranes of the chief cells. (B) Fluorescent confocal images of the pit regionof gastric glands for KRAP (red), IP3R3 (green), and the merged photo. Arrowsindicate the apical membranes of the mucous cells. (C) Fluorescent confocal images ofthe base region of gastric glands for IP3R2 (red), IP3R3 (green),and the merged photo. Asterisks and arrow indicate the parietal cells and the apicalmembranes of the chief cells, respectively. (D) Fluorescent confocal images of the pitregion of gastric glands for IP3R2 (red), IP3R3 (green), and themerged photo. Arrows indicate the apical membranes of the mucous cells. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.
Mentions: Since we previously reported that KRAP associates with particular subtypes ofIP3R in the liver and the pancreas (20), we examined whether KRAP in the stomach is also co-localizedwith IP3R. Double-immunostaining of the stomach for KRAP and IP3R3revealed that KRAP was co-localized with IP3R3 in the apical regions of boththe chief cells (Fig. 2A, arrows) andthe mucous cells (Fig. 2B, arrows).Of note, IP3R2 co-existed with IP3R3 in the chief cells (Fig. 2C, arrow) but not in the parietalcells (Fig. 2C, asterisks).Furthermore, IP3R2 was not detected in the mucous cells (Fig. 2D, arrows). These results indicatedthat KRAP was co-localized with IP3R2 and IP3R3 in the chief cellsand with IP3R3 in the mucous cells.

Bottom Line: We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas.Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs.These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

ABSTRACT
KRAS-induced actin-interacting protein (KRAP), originally identified as one of the deregulated genes expressed in colorectal cancer, participates under physiological conditions in the regulation of systemic energy homeostasis and of the exocrine system. We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas. However, the expression of KRAP and its precise function in other tissues remain elusive. In this study, we aimed to identify the KRAP-expressing cells in mouse stomach and kidneys and to examine the relevance of KRAP expression in the regulation of IP(3)R localization in these tissues. In the stomach, double immunohistochemical staining for KRAP and IP(3)R demonstrated that KRAP was expressed along with the apical regions in the mucous cells and the chief cells, and IP(3)R3 was dominantly co-localized with KRAP in these cells. Furthermore, IP(3)R2 was also co-localized with IP(3)R3 in the chief cells. It is of note that the proper localization of IP(3)R3 and IP(3)R2 in the chief cells and of IP(3)R3 in the mucous cells were significantly abrogated in KRAP-deficient mice. In the kidneys, KRAP was expressed in both the apical and the basal regions of the proximal tubular cells. Intriguingly, KRAP deficiency abrogated the localization of IP(3)R1 in the proximal tubular cells. Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs. These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

Show MeSH
Related in: MedlinePlus