Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction.
The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.
Brandt
N R
NR
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida.
Caswell
A H
AH
Brandt
T
T
Brew
K
K
Mellgren
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology; D002154:Calpain / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009124:Muscle Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; C033668:calpastatin; D000255:Adenosine Triphosphate; D002154:Calpain