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 includes a protein kinase A phosphorylation site. Expressed peptides are DHPr664-799 encoding rabbit skeletal DHPr alpha1 subunit amino acids 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), triadin 2b (242-389), triadin 2b1 (242-299), triadin 3 (370-706), triadin 3a (370-562), triadin 3b (551-706), triadin 3b1 (551-672), and triadin 3b2 (673-706) (the numbers in parentheses correspond to the amino acid sequence of triadin). The triadin monoclonal antibodies, GE4.90 and AE8.91, bind to intact triadic vesicles as well as to vesicle fragments prepared after treatment with Triton X-100, indicating that they have cytoplasmic epitopes. MAb AE8.91 binds to triadin 2, 2', 2a, and 2a1, while mAb GE4.90 binds to triadin 3, 3b, and 3b2 indicating that residues 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 no to triadin 1 or triadin 3b2. The binding to triadin 2a is the most prominent. Direct binding between DHPr-644-799 and JFP was not seen. These experimental findings indicate that triadin contains an extensive cytoplasmic domain that binds to the domain of DHPr which is considered critical for signal transmission during skeletal muscle excitation-contraction sampling.
Fan
H
H
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33136, USA.
Brandt
N R
NR
Peng
M
M
Schwartz
A
A
Caswell
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D000939:Epitopes; D009124:Muscle Proteins; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C070048:triadin