BINDING-SITES OF MONOCLONAL-ANTIBODIES AND DIHYDROPYRIDINE RECEPTOR ALPHA(1) SUBUNIT CYTOPLASMIC II-III LOOP ON SKELETAL-MUSCLE TRIADIN FUSION PEPTIDES

Triadin binds to the dihydropyridine receptor (DHPr) and the junction foot protein (JFP) in Western blot protein overlay experiments. Fusion peptides were synthesized using an expression system, pGSTag, which i ncludes a protein kinase A phosphorylation site. Expressed peptides ar e DHPr664-799 encoding rabbit skeletal DHPr alpha(1) subunit amino aci ds 664-799, triadin 1 (1-49), triadin 2 (68-389), triadin 2' (110-389) , triadin 2a (68-278), triadin 2a1 (67-163), triadin 2a2 (165-240), tr iadin 2b (242-389), triadin 2b1 (242-299), triadin 3 (370-706), triadi n 3a (370-562), triadin 3b (551-706), triadin 3b1 (551-672), and triad in 3b2 (673-706) (the numbers in parentheses correspond to the amino a cid sequence of triadin). The triadin monoclonal antibodies, GE4.90 an d AE8.91, bind to intact triadic vesicles as well as to vesicle fragme nts prepared after treatment with Triton X-100, indicating that they h ave cytoplasmic epitopes. MAb AE8.91 binds to triadin 2, 2', 2a, and 2 a1, while mAb GE4.90 binds to triadin 3, 3b, and 3b2 indicating that r esidues 110-163 and the C-terminal 34 amino acids contain cytoplasmic domains. Radiolabeled DHPr664-799 binds to triadin in intact vesicles under nonreducing and reducing conditions. It binds to triadin fusion peptides, triadin 2, 2a, 3, 3b, and 3b1, but not to triadin 1 or triad in 3b2. The binding to triadin 2a is the most prominent. Direct bindin g between DHPr664-799 and JFP was not seen. These experimental finding s indicate that triadin contains an extensive cytoplasmic domain that binds to the domain of DHPr which is considered critical for signal tr ansmission during skeletal muscle excitation-contraction coupling.