Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

doi:10.1016/0006-291X(82)90887-7

Biochemical and Biophysical Research Communications

Volume 108, Issue 2, 30 September 1982, Pages 708-714

https://doi.org/10.1016/0006-291X(82)90887-7 Get rights and content

Abstract

Mild proteolytic treatment of human platelet membranes by alpha-chymotrypsin increased basal adenylate cyclase activity and abolished the inhibition induced by epinephrine in the presence of GTP. This treatment did not alter the total number of yohimbine binding sites, but markedly decreased the proportion of high affinity, GTP-sensitive sites for epinephrine as assessed from epinephrine competition studies. The effect of proteolysis was dose-dependent upon both adenylate cyclase inhibition and alpha₂-adrenergic binding sites, with a half-maximal effect occurring at similar alpha-chymotrypsin concentration. These results support the concept that only one protein is responsible for the GTP regulation of receptor and inhibition of cyclase.

References (25)

HanouneJ. et al.
J. Biol. Chem.
(1977)
StengelD. et al.
FEBS Lett.
(1980)
YamamuraH. et al.
J. Biol. Chem.
(1977)
AndersonW.B. et al.
Arch. Biochem. Biophys.
(1981)
MarshakD.R. et al.
J. Biol. Chem.
(1980)
PinkettM.O. et al.
J. Biol. Chem.
(1980)
MotulskyH.J. et al.
Biochem. Biophys. Res. Commun.
(1980)
HoffmanB.B. et al.
J. Biol. Chem.
(1980)
LowryO.H. et al.
J. Biol. Chem.
(1951)
WhiteA.A.
Meth. Enzymol.
(1974)

AktoriesK. et al.

FEBS Lett.

(1981)

CooperD.M.F.

FEBS Lett.

(1982)

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Adenylate cyclase in synaptic plasma membranes from rat brain is activated by α-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with α-chymotrypsin (100 μg/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10–30 μg/ml) much more weakly enhances the cyclase activity. (2) α-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not α-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of α-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5′-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified βγ-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the βα-subunits with α-chymotrypsin (> 10 μg/ml). (6) Trypsin (1–10 μg/ml) inactivates the GTPase of the α-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin.
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Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Abstract

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Meth. Enzymol.

FEBS Lett.

FEBS Lett.