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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 interacts with IP3R1 in the kidney and with IP3R3 in thestomach. (A) Western blots showing comparable expression levels of IP3R1 inthe kidney (left) or of IP3R3 in the stomach (right) betweenKRAP-deficient (KO) and wild-type (WT) mice. (B) Anti-KRAP(αKRAP) immunoprecipitations were performed using mouse kidneys and stomachsfrom WT or KO mice, followed by western blotting with anti-KRAP, anti-IP3R1,or anti-IP3R3 antibodies. total, total lysate;IP, immunoprecipitation;α, anti-.
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f5-ijmm-30-06-1287: KRAP interacts with IP3R1 in the kidney and with IP3R3 in thestomach. (A) Western blots showing comparable expression levels of IP3R1 inthe kidney (left) or of IP3R3 in the stomach (right) betweenKRAP-deficient (KO) and wild-type (WT) mice. (B) Anti-KRAP(αKRAP) immunoprecipitations were performed using mouse kidneys and stomachsfrom WT or KO mice, followed by western blotting with anti-KRAP, anti-IP3R1,or anti-IP3R3 antibodies. total, total lysate;IP, immunoprecipitation;α, anti-.

Mentions: As described above, immunohistochemical signals for particular IP3R subtypesin the KRAP-KO mouse kidneys or the stomach were abrogated, leading us tocheck the expression levels of IP3R between the WT and KRAP-KOmouse tissues. Normal expression levels of IP3R1 and IP3R3 weredetected in the KRAP-KO mouse kidney and the stomach, respectively,compared with the WT mouse tissues (Fig.5A), suggesting that mislocalizations but not deregulated expressions ofIP3R occur in the KRAP-KO mouse kidneys and the stomach.Next, to examine the physical association of KRAP with IP3R, we performedco-immunoprecipitations by anti-KRAP antibody in the kidneys or the stomach, in which wecould not evaluate the specific association of IP3R2 with KRAP due to lack ofIP3R2-specific antibody available for western blotting. In the preparationsfrom the WT mouse tissues, KRAP precipitates IP3R1 and IP3R3 in thekidney and the stomach, respectively (Fig.5B). The specificity of co-immunoprecipitations of IP3R was confirmedby using KRAP-KO mouse tissue as a control (Fig. 5B). Thus, KRAP physically interactswith IP3R1 in the kidneys and with IP3R3 in the stomach.


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 interacts with IP3R1 in the kidney and with IP3R3 in thestomach. (A) Western blots showing comparable expression levels of IP3R1 inthe kidney (left) or of IP3R3 in the stomach (right) betweenKRAP-deficient (KO) and wild-type (WT) mice. (B) Anti-KRAP(αKRAP) immunoprecipitations were performed using mouse kidneys and stomachsfrom WT or KO mice, followed by western blotting with anti-KRAP, anti-IP3R1,or anti-IP3R3 antibodies. total, total lysate;IP, immunoprecipitation;α, anti-.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ijmm-30-06-1287: KRAP interacts with IP3R1 in the kidney and with IP3R3 in thestomach. (A) Western blots showing comparable expression levels of IP3R1 inthe kidney (left) or of IP3R3 in the stomach (right) betweenKRAP-deficient (KO) and wild-type (WT) mice. (B) Anti-KRAP(αKRAP) immunoprecipitations were performed using mouse kidneys and stomachsfrom WT or KO mice, followed by western blotting with anti-KRAP, anti-IP3R1,or anti-IP3R3 antibodies. total, total lysate;IP, immunoprecipitation;α, anti-.
Mentions: As described above, immunohistochemical signals for particular IP3R subtypesin the KRAP-KO mouse kidneys or the stomach were abrogated, leading us tocheck the expression levels of IP3R between the WT and KRAP-KOmouse tissues. Normal expression levels of IP3R1 and IP3R3 weredetected in the KRAP-KO mouse kidney and the stomach, respectively,compared with the WT mouse tissues (Fig.5A), suggesting that mislocalizations but not deregulated expressions ofIP3R occur in the KRAP-KO mouse kidneys and the stomach.Next, to examine the physical association of KRAP with IP3R, we performedco-immunoprecipitations by anti-KRAP antibody in the kidneys or the stomach, in which wecould not evaluate the specific association of IP3R2 with KRAP due to lack ofIP3R2-specific antibody available for western blotting. In the preparationsfrom the WT mouse tissues, KRAP precipitates IP3R1 and IP3R3 in thekidney and the stomach, respectively (Fig.5B). The specificity of co-immunoprecipitations of IP3R was confirmedby using KRAP-KO mouse tissue as a control (Fig. 5B). Thus, KRAP physically interactswith IP3R1 in the kidneys and with IP3R3 in the stomach.

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