title: Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP).
abstract: The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.
authors: Department of Physiology, University of Toronto, Ontario M5G 1L7, Canada.
authors: Timothy
authors: T
authors: Ryan
authors: Parveen
authors: P
authors: Sharma
authors: Alex
authors: A
authors: Ignatchenko
authors: David H
authors: DH
authors: MacLennan
authors: Thomas
authors: T
authors: Kislinger
authors: Anthony O
authors: AO
authors: Gramolini
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D037241:Mannose-Binding Lectins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: C410225:CHERP protein, human; D015220:Calcium Channels; D004268:DNA-Binding Proteins; D007093:Imidazoles; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D016601:RNA-Binding Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C029899:imidazole; D010764:Phosphorylase Kinase