Treatment of rat pituitary GH4C1 cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]), but did not attenuate MnCl2 action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of Gsα, but not by anti-Gi2α or anti-Gβ antibodies. Furthermore, it was shown that Gsα is more susceptible to calpain-mediated proteolysis than Gi2α or Gβ. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of Gsα in GH4C1 cell membranes. Proteolysis of Gsα by μ-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH4C1 cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH4C1 cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH4C1 cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of Gsα, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of Gsα to an active Gsα fragment. This is a novel calpain-dependent activation mechanism of Gsα and, thus, of adenylate cyclase in rat pituitary cells.
Research Article| April 25 2000
Persistent activation of Gsα through limited proteolysis by calpain
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Kaori SATO-KUSUBATA, Yukiko YAJIMA, Seiichi KAWASHIMA; Persistent activation of Gsα through limited proteolysis by calpain. Biochem J 1 May 2000; 347 (3): 733–740. doi: https://doi.org/10.1042/bj3470733
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