Journal of Molecular Biology
The Muscle Ankyrin Repeat Proteins: CARP, ankrd2/Arpp and DARP as a Family of Titin Filament-based Stress Response Molecules
Introduction
Striated muscle myofibrils are comprised of intricate networks of cytoskeletal elements and regulatory molecules whose interactions are essential for the ability of different muscle fibers to contract efficiently.1., 2. Importantly, these networks of molecules also allow striated muscle to respond to subtle environmental cues by signaling to induce modifications to their trophic states or fiber type compositions.3., 4. Major components of these networks include the sarcomeres, the basic contractile units of muscle, which are composed of precisely aligned filament systems including the thick (myosin) and thin (actin) filament systems. In addition, vertebrate myofibrils contain a third filament system formed by the giant filamentous titin polypeptide (3000–3700 kDa). The titin filament, which spans entire half-sarcomeres, appears to have many functions. Titin contains a series of spring elements within its I-band region that contribute to the elastic properties of myofibrils. External or internal forces applied to the myofibril (i.e. passive stretch to above the slack length;5., 6. or active contraction to below this length; for further details, see Figure 1 of Granzier & Labeit6) create a titin force, which is directed to restore resting length. By this titin-based mechanism, physiological sarcomere lengths are limited to a narrow range and A-bands are maintained centrally in the sarcomeres. Structurally, titin is important for myofibrillar integrity, since it forms a continuous filament system within myofibrils. Its N-terminal ∼80 kDa region is an integral part of the Z-line, and its C-terminal 200 kDa region is integrated in the M-line lattice. Consequently, titin filaments from adjacent sarcomeres overlap within the Z-line, and titins from adjacent half-sarcomeres overlap within the M-line lattice.7 Thus the many titins contained in series within a myofibril are functionally connected and distribute forces equally between sarcomeres.
In addition to titin's structural and elastic properties, mounting evidence indicates that this giant molecule participates in multiple myofibril signaling pathways. For instance, titin contains a unique Ser/Thr kinase domain in its C-terminal region whose absence in conditional mutant mice leads to sarcomeric disassembly and early death.8 Recent studies on Z-disc and M-line titin regions have also identified an unexpected variety of signaling proteins that interact with titin. Titin's Z-line region (repeats Z1–Z2) interacts with the protein T-cap/telethonin,9., 10. which in turn is predicted to interact with: (1) a minK potassium channel subunit,11 (2) the muscle growth factor myostatin,12 and (3) the muscle LIM protein (MLP).13 Titin Z1–Z2 also interacts with the sarcoplasmic reticulum protein, small ankyrin-1 (sAnk1), suggesting that titin, T-cap/telethonin, and sAnk1 may function in organizing the sarcoplasmic reticulum around myofibrils.14 The titin domain Z4 from titin's central Z-disc region and the recently discovered alternative titin exon novex-3 (located close to the Z-line), both interact with the 700 kDa muscle protein obscurin.15., 16. Obscurin contains PH, DH, IQ and kinase signaling domains,15., 16. and interacts with the ankyrin isoform 1.5; this ankyrin isoform appears to link myofibrils to the sarcoplasmic reticulum and may regulate ryanodine receptor distribution in the sarcoplasmic reticulum.17 Finally, the RING finger protein MURF-1 binds to titin's M-line region; MURF-1 is likely to be involved in the regulation of gene expression and muscle protein assembly and metabolism.18., 19., 20.
What is the function of the multiple titin filament-associated signaling molecules? Structurally, the obscurin epitopes from the Z-line peripheral region increase their distance to the Z-disc by about twofold upon stretch.16 Therefore, it was speculated that the novex-3 titin/obscurin complex participates in myofibrillar stretch-dependent signaling pathways. Similarly, recent evidence suggests that the T-cap/MLP/titin Z1–Z2 complex is part of a stretch-dependent myocardial signaling pathway whose impairment contributes to the pathogenesis of a subset of dilated cardiomyopathies in humans.13 These studies indicate that myofibrillar strain mediates signaling pathways that involve titin's Z-line region.13., 21., 22. Most importantly, a greater understanding of the players that link myocellular stress/strain dependent pathways with trophic/hypertrophic responses should provide us with new therapeutic strategies for treating these diseases.23
The elastic properties of titin's I-band segment make it an ideal candidate for harboring a stretch-dependent myofibril sensor(s). Of particular interest are titin's N2B and N2A segments, which constitute unique linker sequences that separate titin's two principal spring elements, the tandem immunoglobulin (Ig) and PEVK spring regions. For the N2B segment of titin, its association with alpha B-crystallin upon ischemia24 and its phosphorylation after β-adrenergic stimulation25 support its potential signaling role. Here, we studied the functional roles of the titin N2A segment by searching for its ligands. This identified a tyrosine-rich binding motif within the N2A region of titin that specifically interacts with a conserved motif present in the three homologous muscle ankyrin repeat proteins (MARPs): CARP/MARP, ankrd2/Arpp, and DARP. All three molecules were identified previously by their cytokine-like induction following cardiac injury and muscle denervation (CARP/MARP),26., 27., 28. skeletal muscle stretch (ankrd2/Arpp),29 or during recovery after metabolic challenge (DARP).30 Interestingly, excision of the kinase domain from the titin filament via the CreLoxP system causes upregulation of both CARP and ankrd2/Arpp proteins.8
Here, we report that the three MARP proteins are co-expressed in striated muscle tissues and share conserved motifs that interact with titin's N2A segment. The ability of MARPs to bind to titin in adult muscles, to potentially respond to stretch29 and act as nuclear transcriptional regulators26., 27., 28. suggest that MARPs may act as molecular links between myofibrillar stretch-induced signaling pathways and muscle gene expression.
Section snippets
MARP proteins share conserved motifs that interact with titin's elastic N2A region
To search for proteins binding to the titin N2A region, a 1545 bp cDNA fragment encoding titin-I80-I83 (Figure 1(a)) was inserted into BTM117c.31 Screening of ∼1,300,000 clones from a human skeletal muscle cDNA library identified 344 prey clones. Of these, 57 were confirmed in the β-galactosidase assay, and 17 clones were randomly chosen for sequencing. Of the 17 sequenced clones, two clones represented human CARP cDNAs. Nine prey cDNA clones encoded a protein homologous to CARP; these nine
Discussion
Here, we have characterized a family of three structurally homologous ankyrin repeat proteins, the MARPs, that are co-expressed in striated muscles and associate with titin N2A in the central myofibrillar I band region. Immunoelectron microscopy demonstrated that the affinity of the association between MARPs and titin appears to be high enough to persist during myofibrillar stretch (for CARP, Bang et al.36 for ankrd2/Arpp, see Figure 6d). The functional basis for this appears to be the presence
Protein interaction studies
For YTH screens, the human N2A fragment Ig80-Ig83 was amplified from human skeletal muscle cDNA with the primer pair Ig83-S: ttt ctcgagc AAA CCA GCT GTG GCC CCA GCA; Ig83-R: ttt acgcgt ta CAC TGC TCT GAT AGG CAT GGT. The amplified 1545 bp fragment was inserted into BTM117c,31 and the recombinant bait was transformed into Saccharomyces cerevisiae, strain Lc40. For screening, Lc40 cells were co-transformed with 2 μg of amplified human skeletal muscle cDNA, prepared in pGAD10 prey vector (Clontech
Acknowledgements
The authors thank Ryan Mudry for help with microscopy and Figures, Stacy Stanislaw for technical assistance, and Joe Bahl and Yewen Wu for cell isolation. We are indebted to Professor H. Sorimachi for his advice and provision of p94 constructs. This work was supported by the Deutsche Forschungsgemeinschaft (La668/7-1) (to S.L.), the NIH HL062881 (to H.G.) and HL63926 and HL03985 (to C.C.G.), and the American Heart Association 0120586Z (to A.S.M.).
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M.K.M. and M.-L.B. contributed equally to this work.