The receptor for parathyroid hormone and parathyroid hormone-related peptide is hydrolyzed and its signaling properties are altered by directly binding the calpain small subunit.
We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes delta Nt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of delta Nt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.
Shimada
Masako
M
Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA.
Mahon
Matthew J
MJ
Greer
Peter A
PA
Segre
Gino V
GV
D001426:Bacterial Proteins / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004622:Embryo, Mammalian; D005347:Fibroblasts; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C033668:calpastatin; D000242:Cyclic AMP; D005982:Glutathione Transferase; D002154:Calpain; D002118:Calcium