Binding of the N‐terminal 63 kDa portion of connectin/titin to α‐actinin as revealed by the yeast two‐hybrid system

Abstract

Connectin/titin is a 3000 kDa protein which links the myosin filament to the Z-line in vertebrate striated muscle sarcomeres. To search for the Z-line proteins to which connectin binds, the yeast two-hybrid system was applied using cDNA coding the N-terminal 63 kDa fragment of connectin. Two clones coding the C-terminal half region of a-actinin (amino acids, 343-897 and 446-897) were obtained. Enzyme-linked immunosorbent assay clearly demonstrated the interactions of a-actinin and the N-terminal 63 kDa fragment of connectin in vitro. Thus it is concluded that the N-terminal 63 kDa portion of connectin binds to a-actinin in the Z-line of myofibrillar sarcomeres.

Myogenic Stage, Sarcomere Length, and Protease Activity Modulate Localization of Muscle-specific Calpain

“…s-␣-Actinin is one of the major components of Z (37) and has multiple binding molecules, such as ␣-actin (38), the N-terminal Z-repeat region of connectin (15,16), myopalladin (39), FATZ1/calsarcin-2/myozenin-1 (40), calsarcin-3/myozenin-3 (41), and cypher/ZASP/Oracle (42). Our finding of interaction between NS and s-␣-actinin suggests that p94 also contributes to the integrity of Z.…”

“…Connectin also plays an important role as a scaffold for its specific ligands such as sarcomeric-␣-actinin/␣-actinin 2 (s-␣-actinin) 2 (15,16), T-cap/telethonin (17), and muscle-specific RING-finger protein-1 (MURF-1) (18) as well as p94 (19). These proteins interact with connectin at three significant regions in the sarcomeres, namely, Z-bands and N2A and M-line regions, where highly organized protein complexes form.…”

Telethonin and Other New Proteins of the Z‐Disc of Skeletal Muscle

“…However, the CaM domain in muscle is not able to bind calcium. As is described later, several Z-disc proteins have been reported to bind ®-actinin 2, including nebulin (14 ), ALP (15 ), FATZ (16 ), myotilin (17 ), titin (18)(19)(20), and ZASP (21).…”

“…The difference in number of alternatively spliced repeats in titin isoforms has been suggested to affect the number of cross-links between ®-actinin 2 and F-actin laments (22) and hence to cause variation in Z-disc thickness (23). There appear to be two types of binding sites for titin in ®-actinin 2: 1) the C-terminal CaM domain that interacts with several z-repeats of titin (4,19,20); 2) the zq-Z4 region that binds to two spectrin-like repeats (R2 and R3).…”

A 7.5-kb 3′-Terminal cDNA Sequence of Chicken Skeletal Muscle Nebulin Reveals Its Actin Binding Regions

“…A cDNA fragment was obtained as a by-product, when proteins interacting with the N terminal region of chicken skeletal muscle connectin were screened by the yeast two hybrid system (Ohtsuka et al , 1997). This cDNA (N400) turned out to be a fragment of nebulin cDNA.…”