Identification of a Novel Ankyrin Isoform (AnkG190) in Kidney and Lung That Associates with the Plasma Membrane and Binds α-Na,K-ATPase*
- From the Department of Pediatrics, Division of Pediatric Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
Abstract
Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins. Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. We have cloned and characterized a novel 190-kDa member of this family from a rat kidney cDNA library, which we term AnkG190 based on the predicted size and homology with the larger neuronal AnkG isoform. AnkG190 displays a unique 31-residue amino terminus, a repeats domain consisting of 24 repetitive 33-residue motifs, a spectrin binding domain, and a truncated regulatory domain. Probes derived from the unique amino terminus hybridize to an 8-kilobase message exclusively in kidney and lung and specifically to the kidney outer medullary collecting ducts by in situ hybridization. Transfections of Madin-Darby canine kidney and COS-7 epithelial cell lines with a full-length AnkG190 construct result in (a) expression at the lateral plasma membrane, (b) functional assembly with the cytoskeleton, and (c) interaction with at least one membrane protein, the Na,K-ATPase. Two independent Na,K-ATPase binding domains on AnkG190 are demonstrated as follows: one within the distal 12 ankyrin repeats, and a second site within the spectrin binding domain. Thus, ankyrins may interact with integral membrane proteins in a pleiotropic manner that may involve complex tertiary structural determinants.
Footnotes
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↵* This work was supported by National Institutes of Health Grant R29-DK47072 and by a Grant-in-Aid from the American Heart Association, Connecticut Affiliate (to P. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
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↵‡ To whom correspondence should be addressed. Present address: Pediatric Nephrology, Albert Einstein College of Medicine, Montefiore Medical Center, 111 East 210th St., Bronx, NY 10467.
- Abbreviations:
- kb
-
kilobase pair(s)
- MDCK
-
Madin-Darby canine kidney
- PCR
-
polymerase chain reaction
- bp
-
base pair(s)
- PBS
-
phosphate-buffered saline
- DEPC
-
diethylpyrocarbonate
- PAGE
-
polyacrylamide gel electrophoresis
- Pipes
-
1,4-piperazinediethanesulfonic acid.
-
- Received January 16, 1998.
- Revision received June 16, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
