A comparison of the basal degradation of type I InsP3Rs [d-myo-inositol 1,4,5-trisphosphate receptor], measured by pulse–chase analysis or by analysis of immunoreactive InsP3Rs after cycloheximide addition, indicated that the small pool of newly synthesized radioactive InsP3Rs degraded relatively rapidly compared with the large pool of mature InsP3Rs. An antibody (Ab) against a peptide sequence within the IL-3 (third intraluminal loop) of the receptor (IL-3 Ab) was used to identify protected proteolytic fragments that may accumulate in cells. The IL-3 Ab recognized a 56 kDa fragment in both WB rat liver cells and A7R5 smooth-muscle cells. Gel filtration experiments indicated that the 56 kDa fragment was monomeric and, based on reactivity to other Abs, was missing the cytosol-exposed N- and C-terminal segments of the receptor. The addition of the lysosomal protease inhibitor chloroquine resulted in the rapid disappearance of the 56 kDa band. This effect was mimicked by the cysteine protease inhibitors leupeptin, N-acetyl-l-leucyl-l-leucyl-l-methioninal and N-acetyl-leucyl-leucyl-norleucinal. Lactacystin and NH4Cl were less effective. A second fragment of 16 kDa containing the C-terminus accumulated only when the cells were treated with NH4Cl, and not with any of the other inhibitors tested. No N-terminal-reactive fragments were observed. We propose that mature InsP3R tetramers dissociate into monomers and that the 56 kDa fragment is a cleavage intermediate of the monomer representing the six transmembrane domains. Angiotensin-II-stimulated down-regulation of InsP3Rs in WB cells has been shown to involve the ubiquitin/proteasome pathway. Angiotensin-II treatment of WB cells neither resulted in the accumulation of any new fragments nor increased the levels of the 56 or 16 kDa fragments. We conclude that basal and agonist-stimulated degradations of InsP3Rs occur by different pathways. The agonist-mediated pathway involves the concerted removal and proteolysis of the entire receptor molecule from the endoplasmic reticulum membrane without the appearance of intermediate intraluminal fragments.

Abbreviations used: Ab, antibody; ALLM, N-acetyl-l-leucyl-l-leucyl-l-methioninal; ALLN, N-acetyl-leucyl-leucyl-norleucinal; Ang-II, angiotensin II; CFTR, cystic fibrosis transmembrane conductance regulator; CT-1, C-terminal Ab; ER, endoplasmic reticulum; InsP3, d-myo-inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; IL-3, third intraluminal loop; TM, transmembrane.

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