Identification of AnkG107, a muscle-specific ankyrin-G isoform

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 AnkG107, a novel ankyrin-G from rat skeletal muscle. AnkG107, 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 AnkG107. In adult muscle, these polypeptides appeared significantly decreased, and immunofluorescence labeling was no more detectable. Green fluorescent protein-tagged AnkG107 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 AnkG107 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 AnkG107 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.