Interaction of Integrin-linked Kinase with the Kidney Chloride/Bicarbonate Exchanger, kAE1

Abstract

Anion exchanger 1 (AE1, 6 band 3, SLC4A1) is a bicarbonate transporter involved in maintaining acid-base homeostasis in the human body (1). There are two human forms of AE1, erythroid (eAE1) and kidney (kAE1). eAE1 or band 3, is the major integral membrane glycoprotein of the erythrocyte, where it serves dual roles of Cl Ϫ /HCO 3 Ϫ exchange and cytoskeletal anchorage to red cell membranes (2). kAE1 is the basolateral Cl Ϫ /HCO 3 Ϫ exchanger of the acid-secreting ␣-intercalated cell of the kidney distal tubule (3). Transcription of eAE1 in erythroid precursors is under the control of an erythroid-specific promoter upstream of exon 1, whereas renal transcription arises from a distinct promoter within intron 3 of the AE1 gene (4). Thus, the resultant kidney transcript encodes the kAE1 polypeptide lacking 65 amino acids present at the N terminus of human eAE1 (5). This structural alteration causes major functional differences between eAE1 and kAE1. The N terminus of eAE1 interacts with many proteins, including ankyrin, proteins 4.1, and glycolytic enzymes (3, 6), whereas the N terminus of kAE1 does not bind to these proteins (7,8).The three-dimensional structure of the N-terminal 43-kDa cytoplasmic domain of eAE1 revealed a globular structure, composed of 11 ␤-strands and 10 ␣-helical segments arranged as an N-terminal interaction domain and a C-terminal dimerization domain (2), but no structure is available for kAE1. Residues 58 -68 of eAE1 form the first ␤-strand in the cytoplasmic domain. Loss of a central strand of ␤-sheet in kAE1 may thus greatly alter the globular structure of the cytosolic domain, thereby altering its protein interactions. Furthermore, the identity of kAE1-binding protein(s) in ␣-intercalated cells remains unknown. Previously a protein called kanadaptin (kidney anion exchanger adaptor protein) was reported to interact with mouse N-terminal kAE1 but not to eAE1 (9). Human kanadaptin does not, however, interact with human kAE1 and localizes predominantly to the nucleus (10). This leads to the question: * This work was supported in part by Canadian Institutes of Health Research operating grants (to J. R. C. and R. A. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Anion Exchanger 1 Interacts with Nephrin in Podocytes

“…Ϫ/Ϫ and podo-Ilk Ϫ/Ϫ mice, together with previously reported interactions of ILK with AE1 12 and nephrin, 32 strongly suggest that the three molecules may exist in a multiprotein complex. Within this complex, kAE1 may act as a "functional bridge" connecting the signal complexes of the SD with those of the basal domain, by simultaneously binding ILK 12 and nephrin, via its N-and C-terminal domains, respectively.…”

“…There was no podocyte detachment from the GBM. In view of the associations of ILK with kAE1 12 and with nephrin 32 and considering the phenotype of mice with podocyte-specific Ilk deletion (podo-Ilk Ϫ/Ϫ mice), [32][33][34] we examined the possible in vivo interrelationship among the three proteins by confocal immunofluorescence microscopy ( Figure 8). In wild-type mouse glomeruli (Figure 8, A through F), both nephrin and AE1 demonstrated linear patterns of staining along the GBM, consistent with podocyte staining, and each colocalized with ILK.…”

“…In view of the direct associations of ILK with kAE1 12 and nephrin, 32 we investigated the physiologic significance of the nephrin/kAE1 interaction. Our studies demonstrate the importance of nephrin for stable kAE1 expression in podocytes and the in vivo interdependence of levels and subcellular localization among kAE1, nephrin, and ILK in podocytes, suggesting a novel role of kAE1 in glomerular function.…”

“…Both the N-and C-terminal domains of kAE1 contain tyrosine residues critical for basolateral targeting, 10,11 which is likely regulated by phosphorylation. 11 The N-terminus of kAE1 interacts with integrin-linked kinase (ILK), 12 a protein that binds the cytoplasmic domains of ␤-integrins and cytoskeleton-associated proteins. 13,14 The kAE1/ILK interaction enhanced kAE1 trafficking to the plasma membrane in HEK293 cells, 12 but deletion of the majority of the ILKinteracting region in kAE1 did not affect its polarized trafficking in MDCK cells.…”

“…11 The N-terminus of kAE1 interacts with integrin-linked kinase (ILK), 12 a protein that binds the cytoplasmic domains of ␤-integrins and cytoskeleton-associated proteins. 13,14 The kAE1/ILK interaction enhanced kAE1 trafficking to the plasma membrane in HEK293 cells, 12 but deletion of the majority of the ILKinteracting region in kAE1 did not affect its polarized trafficking in MDCK cells. 11 Thus, the physiologic importance of the kAE1-ILK interaction in the kidney remains unclear.…”