Abstract
cDNAs encoding trpE fusion proteins containing fragments of the skeletal muscle Ca2+ release channel (ryanodine receptor) were expressed in bacteria. The fusion proteins, which covered about 90% of the linear sequence of the ryanodine receptor, were used to identify calmodulin- (CaM), Ca(2+)-, and ruthenium red-binding regions in the ryanodine receptor through the use of 125I-CaM, 45Ca2+, and ruthenium red overlay procedures. Six Ca(2+)-dependent CaM-binding domains were detected in the skeletal muscle ryanodine receptor. Strong CaM-binding domains were localized in regions 6, 11, 12, and 13, in subregions 6b, 11b, and 13b, and in short sequences 6b3, 11b1, and 13b2, lying between amino acid residues 2063 and 2091, 3611 and 3642, and 4303 and 4328. Weaker CaM-binding domains were localized in regions 4, 9, and 10 and in subregions 4b, 9b, and 10a, lying between residues 921 and 1173, 2804 and 2930, and 2961 and 3084. Most of these CaM-binding domains encompassed all or part of previously predicted CaM-binding sites. Strong 45Ca(2+)- and ruthenium red-binding sites domains were localized in the NH2- and COOH-terminal regions of the ryanodine receptor and in regions 6, 12, and 13. The 45Ca(2+- and ruthenium red-binding sites in regions 6 and 12 were localized in subregions 6b and 12b, lying between residues 1861-2094 and 3657-3776. These data together with earlier studies (Chen, S. R. W., Zhang, L., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 23318-23326), show that strong CaM-, Ca(2+)-, and ruthenium red-binding domains are colocalized in the skeletal muscle ryanodine receptor.
Highlights
Quence of the ryanodine receptor, were usedto identify encoding ryanodine receptors has provided insight into thelocalmodulin-(CaM), Ca2+-a,nd ruthenium red-bindingre- calization of binding sites for some of these ligands(Takeshima gions in theryanodine receptor through the use of lZSI- et al, 1989;Zorzato et al, 1990; Penner et al, 1989; Chen et al., CaM, 4sCa2,+and ruthenium red overlay procedures
Best Institute, Toronto, Ontario-MSG lL6, Caiada cDNAs encodingtrpE fusion proteins containing frag- bilayers (Smith et at., 1985,1988; Hymel et al, 1988; Lai et al, ments of the skeletal muscle Ca2r+elease channel and of channels expressed in COS-1 cells from a single odine receptor) were expressed in bacteria
A SacI (10175)lBamHI(10971) fragment was ligated into the SacI and BamHI sites in the polylinker in PATH11 to form pFP11.APstI (10646)lPstI(polylinker)from pFPll was removed, 1251-CalmodulinBinding to trpE Fusion Proteins-Synthetic peptides have been widely used in the identification of predicted CaM'-binding sites in various proteins (Chapman et al, and the remaining vector insert was religated to form pFPlla (Fig. 1). 1991; Hofmann et al, 1993; Vorherr et al, 1993), indicating
Summary
BamHI (4893)fragment was ligated into the SacI and BamHI sites in Fusion proteins or sarcoplasmicreticulum proteins were denatured in the polylinker in PATH10 to form pFP5.APstI (6283)lPstI(7423)frag- Laemmli sample buffer at 100 "Cfor 2 min and separated in 8.5%. The remaining vector insert was religated after blunt ending with Klenow to form pFPlOa. A SacI (10175)lBamHI(10971) fragment was ligated into the SacI and BamHI sites in the polylinker in PATH11 to form pFP11.APstI (10646)lPstI(polylinker)from pFPll was removed, 1251-CalmodulinBinding to trpE Fusion Proteins-Synthetic peptides have been widely used in the identification of predicted CaM'-binding sites in various proteins (Chapman et al, and the remaining vector insert was religated to form pFPlla (Fig. 1). Primers 6b2F and 6b2R,correspondingto nucleotides5880-5902 and Tzagoloff, 1985)that contain small fragments of the ryanodine receptor covering about 90%of the ryanodine receptor sequence (Fig. 1).Calmodulin binding to these fusion proteins was examined by an overlay procedure (Flanagan and Yost, 1984).
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