Persistent activation of Gsα through limited proteolysis by calpain

Abstract

Treatment of rat pituitary GH(4)C(1) 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 5'-[gamma-thio]triphosphate (GTP[S]), but did not attenuate MnCl(2) 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 G(s)alpha, but not by anti-G(i)2alpha or anti-Gbeta antibodies. Furthermore, it was shown that G(s)alpha is more susceptible to calpain-mediated proteolysis than G(i)2alpha or Gbeta. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of G(s)alpha in GH(4)C(1) cell membranes. Proteolysis of G(s)alpha by micro-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 GH(4)C(1) cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH(4)C(1) cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH(4)C(1) cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of G(s)alpha, 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 G(s)alpha to an active G(s)alpha fragment. This is a novel calpain-dependent activation mechanism of G(s)alpha and, thus, of adenylate cyclase in rat pituitary cells.

Targeted Proteolysis Sustains Calcineurin Activation

“…18 Therefore, calpainmediated proteolysis emerges as a powerful activator of the CnA/NF-ATc pathway. Additionally, targeted proteolysis of specific signaling molecules like protein kinase C 19 and of G-proteins 20 by calpain could selectively increase the effect of signaling cascades. The major cardiac calpain isoforms are -calpain (calpain 1) and m-calpain (calpain 2), named according to Ca 2ϩ concentrations for maximum in vitro activity.…”

Conventional calpains and programmed cell death.

“…Other well documented examples are regulation of adhesion complexes by calpain, that is required for cell migration and motility, and calpain-mediated NMDA receptor truncation believed to be a key factor for feed-back regulation of the receptor activity (Bi et al, 1998;Franco et al, 2004;Liu et al, 2005). There is also growing evidence for an important role of calpain in signal transduction via the generation of cAMP through G-protein-coupled receptors by proteolytic activation of Gs α (Sato- Kusubata et al, 2000). Removal of the N-terminal region from tyrosine hydroxylase by calpain, resulting in disruption of the quaternary structure, changes its susceptibility to physiological regulatory mechanisms (Kiuchi et al, 1991).…”

Endoproteolytic Processing of Sst2, a Multidomain Regulator of G Protein Signaling in Yeast

“…Only the receptor-bound form of arrestin is a substrate, and once proteolyzed it is less able to dissociate from the receptor, apparently leading to prolonged receptor desensitization (56). Calpain has also been shown to cleave the G s ␣ subunit, leading to persistent activation of adenylyl cyclase (57).…”