Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein G_i

Abstract

The antiserum AS7 can specifically immunopreeipitate a-Gi from membrane extracts as well as from a mixture of purified ct-Gi and a-Go as ascertained using [azP]ADP-ribosylated G-proteins. Using this antiserum to immunopreeipitate ~t-G~ from hepatocytes labelled with a2p it was evident that ~t-Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of ~t-Gi. This was accompanied by the loss of inhibitory effect of G~ on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH-glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD +-dependent, [azP]ADP-ribosylation of a-Ga in membrane fractions isolated from such pre-treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of ~t-G~ in intact hepatoeytes. As pertussis toxin only causes the ADP-ribosylation of the holomeric form of G~, it may be that phosphorylation leaves ct-Gi in its holomeric state.

Activation of protein kinase C inhibits hormonal stimulation of the GTPase activity of Gi in human platelets

“…It is in. tercstinB to note that it has been shown that TPA blocks 0i function in liver cells [201 and that this effect is associated with the phosphorylation of Gi or [20]; however, the blockade of Gi function observed in liver cells is differer~t to that observed in platele(s, i,e. the inhibitory action of Ilydrolysis-resistant analogues of GTP is blocked [20], Aeknowledllemenl,t; The authors thank Ms Guadalu p= gamirex for typinll the manuscript, This researcll was partially supported by a grant rrom DGAPA (1N021889),…”

The role of G proteins in transmembrane signalling

“…Phosphorylation of G proteins by, for example, protein kinases C1C. W.Taylor C (Sagi-Eisenberg, 1989: Pyne et al, 1989 or receptor tyrosine kinases (Zick et al, 1986;Valentine-Braun et al, 1986;O'Brien et al, 1987) (Jakobs et al, 1985).…”

Muscarinic blockade of β‐adrenoceptor‐stimulated adenylyl cyclase: the role of stimulatory and inhibitory guanine‐nucleotide binding regulatory proteins (Gs and Gi)

“…Pertussis toxin-catalysed ADP-ribosylation was performed according to Pyne et al (1989). Membranes (10Ltg) were combined with a final concentration (mM): thymidine 30, potasium phosphate 80 (pH 7.5), arginine 20, MgCl2 1 and to this was added 2,Ci [32P]-NAD' 0.02, and these were then combined with preactivated pertussis toxin (0.64 pLg/assay) and incubated at 37°C for 30 min.…”

“…This is a typical result from the experiment performed at contrast, methacholine pretreatment of tracheal smooth least 10 times on different membrane preparations. (Katada et al, 1985;Zick et al, 1986;O'Brien et al, 1987;Pyne et al, 1989;1992a) and adenylyl cyclase (Yoshimasa et al, 1987). The molecular events that govern the functional antagonism between the muscarinic receptor and the adrenoceptor Llysed are unknown.…”

“…receptors (Katada et al, 1985;Pyne et al, 1989 catalyse ADP-ribosylation of G. (Figure 8). GppNHp (100 atM) stimulated the ability of G. to act as a substrate for cholera toxin-catalysed ADP-ribosylation in membranes from untreated tracheal smooth muscle.…”