CARP interacts with titin at a unique helical N2A sequence and at the domain Ig81 to form a structured complex

Abstract

The cardiac ankyrin repeat protein (CARP) is up-regulated in the myocardium during cardiovascular disease and in response to mechanical or toxic stress. Stress-induced CARP interacts with the N2A spring region of the titin filament to modulate muscle compliance. We characterize the interaction between CARP and titin-N2A and show that the binding site in titin spans the dual domain UN2A-Ig81. We find that the unique sequence UN2A is not structurally disordered, but that it has a stable, elongated a-helical fold that possibly acts as a constant force spring. Our findings portray CARP/titin-N2A as a structured node and help to rationalize the molecular basis of CARP mechanosensing in the sarcomeric I-band.

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

“…The N2A region of titin plays a role in a number of important binding interactions but little is known about the properties of the individual domains. This is important to understand since different regions in N2A have unique interactions that could be regulated, in part, by domain stability.…”

“…The N2A region is believed to play a variety of crucial roles in both cardiac and skeletal muscle and is required for normal muscle function . The signaling and regulatory functions of the N2A region require functional binding sites for both the muscle ankyrin repeat proteins (MARPs) and the p94 protease.…”

“…The MARP proteins are transcription factors that regulate stress and damage response in both cardiac and skeletal muscle . CARP, the cardiac muscle member of the family, interacts with titin through a binding site that spans part of the insertion sequence and the I81 domain in response to various types of stress signals . The functional significance of the CARP/titin interaction is still not clear but proposed functions include modulation of titin elasticity, protection from phosphorylation and sequestration of CARP to downregulate its transcriptional activity .…”

“…In cardiac muscle, there are two isoforms and the longer of the two titin isoforms (N2BA) includes the N2A region . One important interaction in this region is titin's stretch‐sensing response through binding of CARP (cardiac ankyrin repeat protein) to the insertion sequence and I81 domains in titin's N2A region . CARP binds to the N2A region after being upregulated in response to mechanical and chemical cardiovascular stress …”

Titin‐based mechanosensing modulates muscle hypertrophy

“…As following UDD MARP1 shows no evidence of nuclear localization (Figure 6A–F), therefore interaction with GATA4 is unlikely. However, by binding to titin's N2A region (Figure 6G–P), MARP1 might increase titin‐based stiffness, cross‐link titin filaments12, 49 to sterically hinder signalling, or even form a scaffold for other binding proteins to facilitate N2A‐based hypertrophy signalling. Binding of MARP1 to titin's N2A segment might be facilitated by the differential upregulation of CAPN3, which in its active state (autolytic fragments) is capable of cleaving MARP1, thereby increasing its binding affinity to titin N2A 9.…”