Persistent activation of Gsalpha through limited proteolysis by calpain.
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.
Sato-Kusubata
K
K
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. k-kusu@rinshoken.or.jp
Yajima
Y
Y
Kawashima
S
S
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002772:Cholera Toxin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
C495403:Adcyap1 protein, rat; D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; C400241:benzyloxycarbonylleucyl-leucine aldehyde; D002772:Cholera Toxin; D000242:Cyclic AMP; D002154:Calpain; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases