Degradation of acetylcholine receptors in muscle cells: effect of leupeptin on turnover rate, intracellular pool sizes, and receptor properties.
The cellular mechanisms of degradation of a transmembrane protein, the acetylcholine receptor (AChR), have been examined in a mouse muscle cell line, BC3H-1. The halftime of degradation of cell surface receptors labeled with [125I] alpha-Bungarotoxin ([125I] alpha-BuTx) is 11-16 h. Leupeptin, a lysosomal protease inhibitor, slows the degradation rate two- to sixfold, depending on the concentration of inhibitor used. The inhibition is reversible since the normal degradation rate is regained within 20 h after removal of the inhibitor. Cells incubated with leupeptin accumulate AChR. Little change in the number of surface AChR occurs but the amount of intracellular AChR increases two- to threefold. Accumulated AChR are unable to bind [125I] alpha-BuTx if excess, unlabeled alpha-BuTx is present in the culture medium during leupeptin treatment. Thus, leupeptin causes the accumulation of a surface-derived receptor population not previously described in these cells. Subcellular fractionation studies utilizing Percoll and metrizamide gradient centrifugation in addition to molecular exclusion chromatography suggest that the accumulated AChR reside in a compartment with lysosomal characteristics. In contrast, the subcellular component containing another intracellular pool of AChR not derived from the surface is clearly separated from lysosomes on Percoll gradients. The sedimentation properties of AChR solubilized from the plasma membrane and the lysosomal fraction have been compared. The plasma membrane AChR exhibits a sedimentation coefficient of 9S in sucrose gradients containing Triton, whereas the AChR derived from the lysosomal fraction exists in part in a high molecular weight form. The large aggregate and the organelle in which it resides may represent important intermediates in the degradative pathway of this membrane protein.
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D007976:Leupeptins / Q000494:pharmacology*; D051379:Mice; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D011950:Receptors, Cholinergic / Q000378:metabolism*
D002038:Bungarotoxins; D007976:Leupeptins; D009842:Oligopeptides; D011950:Receptors, Cholinergic; C032854:leupeptin