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PMID:19088451

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Citation

Zachos, NC, Hodson, C, Kovbasnjuk, O, Li, X, Thelin, WR, Cha, B, Milgram, S and Donowitz, M (2008) Elevated intracellular calcium stimulates NHE3 activity by an IKEPP (NHERF4) dependent mechanism. Cell. Physiol. Biochem. 22:693-704

Abstract

The ileal brush border (BB) contains four evolutionarily related multi-PDZ domain proteins including NHERF1, NHERF2, PDZK1 (NHERF3) and IKEPP (NHERF4). Why multiple related PDZ proteins are in a similar location in the same cell is unknown. However, some specificity in regulation of NHE3 activity has been identified. For example, elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2 but not NHERF1, and involves the formation of large NHE3 complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether the four PDZ domain containing protein IKEPP reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, IKEPP bound to the F2 region (aa 590-667) of NHE3 in overlay assays, which is the same region where NHERF1 and NHERF2 bind. PS120 cells lack endogenous NHE3 and IKEPP. Treatment of PS120/NHE3/IKEPP cells (stably transfected with NHE3 and IKEPP) with the Ca(2+) ionophore, 4-Br-A23187 (0.5 microM), stimulated NHE3 V(max) activity by approximately 40%. This was associated with an increase in plasma membrane expression of NHE3 by a similar amount. NHE3 activity and surface expression were unaffected by A23187 in PS120/NHE3 cells lacking IKEPP. Based on sucrose density gradient centrifugation, IKEPP was also shown to exist in large complexes, some of which overlap in size with NHE3, and the size of both NHE3 and IKEPP complexes decreased in parallel after [Ca(2+)](i) elevation. FRET experiments on fixed cells demonstrated that IKEPP and NHE3 directly associated at an intracellular site. Elevating [Ca(2+)](i) decreased this intracellular NHE3 and IKEPP association. In summary: (1) In the presence of IKEPP, elevated [Ca(2+)](i) stimulates NHE3 activity. This was associated with increased expression of NHE3 in the plasma membrane as well as a shift to smaller sizes of NHE3 and IKEPP containing complexes. (2) IKEPP directly binds NHE3 at its F2 C-terminal domain and directly associates with NHE3 in vivo (FRET). (3) Elevated [Ca(2+)](i) decreased the association of IKEPP and NHE3 in an intracellular compartment. Based on which NHERF family member is expressed in PS120 cells, elevated [Ca(2+)](i) stimulates (IKEPP), inhibits (NHERF2) or does not affect (NHERF1) NHE3 activity. This demonstrates that regulation of NHE3 depends on the nature of the NHERF family member associating with NHE3 and the accompanying NHE3 complexes.

Links

PubMed Online version:10.1159/000185553

Keywords

Animals; Calcium/metabolism; Endocytosis; Endosomes/metabolism; Enterocytes/cytology; Enterocytes/metabolism; Fluorescence Resonance Energy Transfer; Humans; Intracellular Space/metabolism; Mice; Mice, Inbred C57BL; Phosphoproteins/metabolism; Protein Binding; Protein Transport; Rabbits; Sodium-Hydrogen Antiporter/metabolism; rab GTP-Binding Proteins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RABIT:SL9A3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q86UT5

F

Seeded From UniProt

complete

RABIT:SL9A3

enables

GO:0015385: sodium:proton antiporter activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

RABIT:SL9A3

located_in

GO:0005903: brush border

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

RABIT:SL9A3

located_in

GO:0009986: cell surface

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:SL9A3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q86UT5

F

Seeded From UniProt

complete

HUMAN:NHRF4

enables

GO:0008022: protein C-terminus binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P48764

F

Seeded From UniProt

complete

HUMAN:NHRF4

located_in

GO:0043296: apical junction complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NHRF4

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P26432

F

Seeded From UniProt

complete

HUMAN:NHRF4

located_in

GO:0045177: apical part of cell

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NHRF4

located_in

GO:0005903: brush border

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:NHRF4

located_in

GO:0005903: brush border

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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