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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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KRAP expression and its contribution to the localization of IP3R1 in theproximal tubules of the mouse kidney. (A and B) Fluorescent confocal images of theproximal tubules of kidney for KRAP (red), F-actin (green), and the merged photo fromwild-type (WT) (A) or KRAP-deficient (KO) (B) mice. Arrowheads andarrows indicate the basolateral and the apical regions of the proximal tubules,respectively. (C and D) Fluorescent confocal images of the proximal tubules of kidneyfor IP3R1 (green), F-actin (red), and the merged photo from WT (C) or KO (D)mice. Blue, 4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.
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f4-ijmm-30-06-1287: KRAP expression and its contribution to the localization of IP3R1 in theproximal tubules of the mouse kidney. (A and B) Fluorescent confocal images of theproximal tubules of kidney for KRAP (red), F-actin (green), and the merged photo fromwild-type (WT) (A) or KRAP-deficient (KO) (B) mice. Arrowheads andarrows indicate the basolateral and the apical regions of the proximal tubules,respectively. (C and D) Fluorescent confocal images of the proximal tubules of kidneyfor IP3R1 (green), F-actin (red), and the merged photo from WT (C) or KO (D)mice. Blue, 4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.

Mentions: To examine the cellular distribution of KRAP protein in the adult mouse kidneys, weperformed immunohistochemical staining by using anti-KRAP antibody. The specificities ofthe signals were validated by comparing the immunoreactivities of WT andKRAP-KO mouse tissues. In the WT kidneys, intense immunoreactivitieswere observed in the renal proximal tubules (Fig. 4A) but not in the renal distal tubules (data not shown). On the otherhand, significant immunoreactive signal was not detected in the proximal tubules in theKRAP-KO mice (Fig.4B). Taken together, these results indicate that KRAP was exactly expressed inthe proximal tubules. The proximal tubules were identified by the presence of thebrush-border stained with phalloidin (Fig.4A and B). Immunostaining in the proximal region showed that KRAP was accumulatedbeneath the brush-border (Fig. 4A,arrows) and KRAP was also detected in the basolateral actin bundles (Fig. 4A, arrowheads). We next examinedwhich subtypes of IP3R, IP3R1, IP3R2, andIP3R3, expressed in the proximal tubular cells, revealing that IP3R1(Fig. 4C) but not IP3R 2or IP3R3 (data not shown) was detected in the beneath the brush-border and inthe basolateral actin bundles. Finally, we addressed the functional relevance of KRAPexpression in the proximal tubular cells to the regulation of IP3Rlocalization. It is of note that the restricted localization of IP3R1 detectedin the WT mouse kidney (Fig. 4C) wasdisturbed in the KRAP-KO mouse kidney (Fig. 4D). Thus, KRAP plays critical role in the regulation of theproper localization of IP3R1 in the proximal tubular 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)

KRAP expression and its contribution to the localization of IP3R1 in theproximal tubules of the mouse kidney. (A and B) Fluorescent confocal images of theproximal tubules of kidney for KRAP (red), F-actin (green), and the merged photo fromwild-type (WT) (A) or KRAP-deficient (KO) (B) mice. Arrowheads andarrows indicate the basolateral and the apical regions of the proximal tubules,respectively. (C and D) Fluorescent confocal images of the proximal tubules of kidneyfor IP3R1 (green), F-actin (red), and the merged photo from WT (C) or KO (D)mice. 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

f4-ijmm-30-06-1287: KRAP expression and its contribution to the localization of IP3R1 in theproximal tubules of the mouse kidney. (A and B) Fluorescent confocal images of theproximal tubules of kidney for KRAP (red), F-actin (green), and the merged photo fromwild-type (WT) (A) or KRAP-deficient (KO) (B) mice. Arrowheads andarrows indicate the basolateral and the apical regions of the proximal tubules,respectively. (C and D) Fluorescent confocal images of the proximal tubules of kidneyfor IP3R1 (green), F-actin (red), and the merged photo from WT (C) or KO (D)mice. Blue, 4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 25μm.
Mentions: To examine the cellular distribution of KRAP protein in the adult mouse kidneys, weperformed immunohistochemical staining by using anti-KRAP antibody. The specificities ofthe signals were validated by comparing the immunoreactivities of WT andKRAP-KO mouse tissues. In the WT kidneys, intense immunoreactivitieswere observed in the renal proximal tubules (Fig. 4A) but not in the renal distal tubules (data not shown). On the otherhand, significant immunoreactive signal was not detected in the proximal tubules in theKRAP-KO mice (Fig.4B). Taken together, these results indicate that KRAP was exactly expressed inthe proximal tubules. The proximal tubules were identified by the presence of thebrush-border stained with phalloidin (Fig.4A and B). Immunostaining in the proximal region showed that KRAP was accumulatedbeneath the brush-border (Fig. 4A,arrows) and KRAP was also detected in the basolateral actin bundles (Fig. 4A, arrowheads). We next examinedwhich subtypes of IP3R, IP3R1, IP3R2, andIP3R3, expressed in the proximal tubular cells, revealing that IP3R1(Fig. 4C) but not IP3R 2or IP3R3 (data not shown) was detected in the beneath the brush-border and inthe basolateral actin bundles. Finally, we addressed the functional relevance of KRAPexpression in the proximal tubular cells to the regulation of IP3Rlocalization. It is of note that the restricted localization of IP3R1 detectedin the WT mouse kidney (Fig. 4C) wasdisturbed in the KRAP-KO mouse kidney (Fig. 4D). Thus, KRAP plays critical role in the regulation of theproper localization of IP3R1 in the proximal tubular 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