Abstract
We previously showed that alternatively spliced ankyrins-G, the Ank3 gene products, are expressed in skeletal muscle and localize to the postsynaptic folds and to the sarcoplasmic reticulum. Here we report the molecular cloning, tissue expression, and subcellular targeting of Ank(G107), a novel ankyrin-G from rat skeletal muscle. Ank(G107) lacks the entire ANK repeat domain and contains a 76-residue sequence near the COOH terminus. This sequence shares homology with COOH-terminal sequences of ankyrins-R and ankyrins-B, including the muscle-specific skAnk1. Despite widespread tissue expression of Ank3, the 76-residue sequence is predominantly detected in transcripts of skeletal muscle and heart, including both major 8- and 5.6-kb mRNAs of skeletal muscle. In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and cross-reacted with three endogenous ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitro translated Ank(G107). In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged Ank(G107) transfected in primary cultures of rat myotubes was targeted to the plasma membrane. Deletion of the 76-residue insert resulted in additional cytoplasmic labeling suggestive of a reduced stability of Ank(G107) at the membrane. Recruitment of the COOH-terminal domain to the membrane was much less efficient but still possible only in the presence of the 76-residue insert. We conclude that the 76-residue sequence contributes to the localization and is essential to the stabilization of Ank(G107) at the membrane. These results suggest that tissue-dependent and developmentally regulated alternative processing of ankyrins generates isoforms with distinct sequences, potentially involved in specific protein-protein interactions during differentiation of the sarcolemma and, in particular, of the postsynaptic membrane.
Highlights
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AJ428573
In 15-day-old rat skeletal muscle, antibodies against the 76-residue sequence localized to the sarcolemma and to the postsynaptic membrane and crossreacted with three endogenous ankyrins-G, including one 130-kDa polypeptide that comigrated with in vitro translated AnkG107
Isolation and Characterization of AnkG107, a Novel Ankyrin-G Isoform from Rat Skeletal Muscle—Ank3 cDNA sequences from the spectrin-binding and COOH-terminal domains were previously amplified by RT-PCR from rat skeletal muscle total RNA [23]
Summary
Sarcolemma (210 kDa) [22, 23, 27] Postsynaptic membrane [23] (isoform ?) Sarcoplasmic reticulum (skAnk1) [22]. These data and the diversity of ankyrin gene expression and localization suggest that ankyrins play key roles in the assembly and functioning of membrane domains in skeletal muscle fibers Most of these isoforms have not yet been identified at the molecular level. Transfection of GFP1-tagged constructs expressing either the full-length molecule or AnkG107 domains in rat myotubes in culture showed that this isoform is targeted to the plasma membrane apparently via the spectrin-binding domain. These experiments strongly suggested that the muscle-
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