Obscurin regulates ankyrin macromolecular complex formation

https://doi.org/10.1016/j.yjmcc.2022.04.008Get rights and content

Highlights

  • AnkG107 and sAnk1.5 bind obscurin via independent binding sites.

  • Obscurin forms a complex with AnkG107 and sAnk1.5 in vitro and in cells.

  • AnkG107 recruits β-spectrin to a complex of obscurin, sAnk1.5, and AnkG107.

  • Removing AnkG107's first obscurin-binding domain disrupts obscurin interaction.

  • AnkG107 obscurin-binding domains do not interact with filamin-C and minimally with plectin-1.

Abstract

Obscurin is a large scaffolding protein in striated muscle that maintains sarcolemmal integrity and aligns the sarcoplasmic reticulum with the underlying contractile machinery. Ankyrins are a family of adaptor proteins with some isoforms that interact with obscurin. Previous studies have examined obscurin interacting with individual ankyrins. In this study, we demonstrate that two different ankyrins interact with obscurin's carboxyl terminus via independent ankyrin-binding domains (ABDs). Using in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis, we show that obscurin interacts with small ankyrin 1.5 (sAnk1.5) and the muscle-specific ankyrin-G isoform (AnkG107). While there is no direct interaction between sAnk1.5 and AnkG107, obscurin connects the two ankyrins both in vitro and in cells. Moreover, AnkG107 recruits β-spectrin to this macromolecular protein complex and mutating obscurin's ABDs disrupts complex formation. To further characterize AnkG107 interaction with obscurin, we measure obscurin-binding to different AnkG107 isoforms expressed in the heart and find that the first obscurin-binding domain in AnkG107 principally mediates this interaction. We also find that AnkG107 does not bind to filamin-C and displays minimal binding to plectin-1 compared to obscurin. Finally, both sAnk1.5-GFP and AnkG107-CTD-RFP are targeted to the M-lines of ventricular cardiomyocytes and mutating their obscurin-binding domains disrupts the M-line localization of these ankyrin constructs. Altogether, these findings support a model in which obscurin can interact via independent binding domains with two different ankyrin protein complexes to target them to the sarcomeric M-line of ventricular cardiomyocytes.

Introduction

Cardiomyocyte contraction is a tightly regulated process that involves the coordinated assembly of cytoskeletal and scaffolding proteins of the sarcomere and internal organelles that regulate calcium cycling. Adaptor proteins such as ankyrins and cytoskeletal proteins such as obscurin play pivotal roles in guiding the incorporation of myofilament and accessory proteins into striated structures and contribute to the alignment of the sarcoplasmic reticulum (SR) and transverse tubules (T-tubules). How these cytoskeletal and cytoskeletal-associated proteins contribute to the subcellular organization of the cardiomyocyte provides valuable information about normal cardiomyocyte function as well as dysfunction in disease.

Obscurins, encoded by the gene OBSCN, are a family of sarcomeric structural and signaling proteins localized to Z-lines and M-lines of myocytes [[1], [2], [3]]. Several mutations in OBSCN have been linked to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), left ventricular noncompaction cardiomyopathy (LVNC), heart failure, and skeletal myopathies [[4], [5], [6], [7]]. The differential subcellular localization of obscurins combined with its numerous modular adhesion and signaling motifs enable it to play pivotal roles in the organization of the sarcomere and maintenance of A-bands and M-lines [1,[8], [9], [10], [11], [12], [13]], in the organization of costameres to withstand stress and transmit contractile force across the sarcolemma [14], and in the organization and cytoskeletal anchoring of the SR membranes [[15], [16], [17], [18], [19]]. One difference between the obscurins found at the M-line, or obscurin-A, versus the Z-line is the presence of two independent ankyrin binding domains in the carboxyl terminus of obscurin-A [20].

Ankyrins are versatile adaptor proteins that facilitate the recruitment and maintenance of protein complexes to their distinct subcellular domains by tethering the integral membrane proteins and signaling molecules to the underlying β-spectrin cytoskeleton. Three genes encode ankyrin proteins (ANK1: ankyrin-R or AnkR, ANK2: ankyrin-B or AnkB, ANK3: ankyrin-G or AnkG) and alternative splicing of these genes yields several modular isoforms [21]. Mutations in ankyrins are linked to several arrhythmias including atrial fibrillation, sinus node dysfunction, long QT syndrome (LQTS), ventricular tachycardia, and Brugada syndrome [[22], [23], [24], [25]]. Furthermore, recent studies show that ankyrin dysfunction has also been associated with arrhythmogenic cardiomyopathy (ACM), DCM, and HCM [[26], [27], [28]].

A canonical ankyrin is composed of four functional domains: the membrane-binding domain (MBD) that binds integral membrane proteins, spectrin-binding domain (SBD) that tethers protein complexes to the cytoskeleton, death domain (DD), and C-terminal domain (CTD). The DD and CTD comprise a regulatory domain because an intramolecular interaction with the MBD regulates MBD and SBD binding activities [29,30]. While the MBD, SBD, and DD share amino acid homology, the CTD shows sequence diversity, conferring unique regulatory mechanisms to each ankyrin protein [30,31]. Within the CTD of many muscle-specific ankyrin isoforms, there reside obscurin binding domains (OBD) that mediate interaction with obscurin [19,[32], [33], [34], [35]].

Many mechanisms contribute to the appropriate subcellular localization of ankyrin protein complexes including interactions with cell adhesion molecules, cytoskeletal proteins, and/or large scaffolding proteins. This paper is the first to investigate whether two independent ankyrin binding domains in the carboxyl terminus of obscurin-A can interact with two different ankyrins in vitro and in cells. Specifically, we examined obscurin interaction with sAnk1.5 and AnkG107. Small Ank1.5 is a ~ 20 kD muscle-specific ankyrin-R isoform, which lacks the MBD and SBD but has a unique transmembrane domain tethering it to the sarcoplasmic reticulum [17,[36], [37], [38]]. Obscurin interaction with sAnk1.5 is important to maintain the longitudinal sarcoplasmic reticulum (lSR) across the contractile apparatus to facilitate uniform calcium release and re-uptake into the SR [18,19,39,40]. AnkG107 is a muscle-specific ankyrin-G isoform that lacks the MBD but has two putative OBDs in its C-terminal domain. While it has been shown that AnkG107 interacts with obscurin by yeast two-hybrid analysis, it has also been reported that the OBDs mediate interaction with plectin-1 and filamin-C [41].

This paper is the first to demonstrate that obscurin can interact with two different ankyrins at the same time. This observation is validated by in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis. Specifically, we demonstrate that sAnk1.5 and AnkG107, which do not interact with each other, are brought together in a complex via the two ankyrin-binding domains in obscurin. We also find that AnkG107 binding obscurin is principally mediated by the first OBD, and that the OBDs do not bind filamin-C and display minimal binding to plectin-1 compared to obscurin. In addition, we show that AnkG107 recruits β1-spectrin to a complex of obscurin and sAnk1.5. Finally, we demonstrate that virally-expressed sAnk1.5 and AnkG107-CTD localize to the M-lines of cardiomyocytes and this localization is disrupted when the obscurin-binding domains are mutated. Altogether, this work characterizes a novel mechanism by which the large scaffolding protein obscurin coalesces two distinct ankyrin protein complexes to the same domain through interactions with its independent ankyrin-binding domains.

Section snippets

Plasmids and antibodies

Fig. 1 contains a diagram of all constructs used in experiments for this paper. Human sAnk1.5 (AF005213, amino acids 29–155) was subcloned in frame with various carboxyl-terminal epitope tags including FLAG, HA, YFP, CFP, and GFP. Human obscurin (NM_052843, amino acids 6148–6460) was subcloned in frame with an amino-terminal GST epitope tag or with various carboxyl-terminal epitope tags including HA, CFP, and GST. To generate ankyrin-binding domain mutants of obscurin (ΔABD1, ΔABD2, ΔABD1&2),

AnkG107/130-CTD binds obscurin

Previous studies have shown that skeletal muscles express different ankyrin-G isoforms including AnkG107 and AnkG130 [[44], [45], [46], [47]]. The mRNA transcripts encoding these isoforms have an alternative start site in Ank3 exon 25 and include the muscle-specific exons 46–49 [44,[46], [47], [48]]. As a result, these isoforms are similar in that they lack membrane-binding domains but express two obscurin-binding domains in their C-termini (Fig. 2A). In contrast, they are different in that

Discussion

There are many mechanisms that regulate ankyrin-mediated protein complex formation, of which the predominant mechanism is the generation of unique alternatively spliced ankyrin isoforms that interact with distinct cohorts of proteins, targeting these complexes to specific subcellular domains. Several studies have shown that alternative splicing of the three ANK genes results in various tissue-specific, modular isoforms that bind discrete sets of proteins [19,29,[34], [35], [36], [37],44,[46],

Accession codes

sAnk1.5AF005213 (NCBI)
ankG107AJ428573 (GenBank)
obscurinNM_052843 (NCBI)
sarcolipinQ6SLE7.1 (Uniprot)
plectin-1NM_011117 (NCBI)
filamin-CNM_001081185 (NCBI)
β1-spectrinJ05500.1 (GenBank)

Author contributions

JS and GY performed the experiments. JS and SRC wrote the paper.

Funding

This work was supported by the American Heart Association (16GRNT30410011).

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgements

Fluorescence microscopy and image analysis was performed at the Nikon Center of Excellence - Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology at McGovern Medical School, UTHealth Houston.

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