Regulation of the phosphorylation state of Gi2 in intact rat hepatocytes

Abstract

Hormonal stimulation and inhibition of intracellular signalling systems such as adenylate cyclase, phospholipases and ion channels require the activation of specific guanine nucleotide-binding G-proteins [ I ] . In addition to regulation by occupied hormone receptors, certain G-proteins may be partially activated, in the absence of extracellular signals, as a consequence of the high intracellular GTP concentrations ( 600 ,LAM). Thus, such G-proteins will exert a tonic stimulatory or inhibitory effect upon their target effector systems. G-proteins are also key sites for ‘cross-talk between the different signalling pathways. This can result in the phenomena of desensitization and of potentiation of specific signalling systems. Such ‘cross-talk’ represents an important mechanism for the control of cellular responsiveness and may be mediated by hormone-induced G-protein phosphorylation [2, 31. Using intact rat hepatocytes, we have examined hormonal regulation of phosphorylation of the G-proteins which control the activity of adenylate cyclase. Hepatocytes express both the G,2 and G,3 forms of the ‘Gi-family’ of G-proteins, but not G, 1. At least one of these G-proteins is believed to couple hormone receptors to the inhibition of adenylate cyclase. We have previously described the production, characterization and use of anti-peptide antisera which selectively recognize the a-subunits of various G-proteins [4]. These anti-peptide antisera were used to immunoprecipitate, selectively, either G,2 (antiserum AS 7), G,3 (antiserum 13B) or G, (antiserum CS1) and their subunits were resolved by polyacrylamide-gel electrophoresis. Using this immunoprecipitation procedure, we found that treatment of intact, 32P-labelled rat hepatocytes with glucagon ( 10 nM, 5 min), TH-glucagon (10 nM, 5 min), arginine-vasopressin (3 nM, S min), angiotensin II (3 nM, 5 min) and the horbol ester 12-0-tetradecanoyl phorbol 13acetate (TPAp( 10 n M , 15 min) elicited 2-2.5-fold maximal increases in the labelling of the a-subunit of immunoprecipitated G12.8-Bromo cyclic AMP (100 ,LAM, 15 min) and higher concentrations of glucagon (1 p ~ , 5 min) which act, at least in part, by activation of A-kinase caused 3.3-4-fold increases (Fig. 1). The effect of 8-bromo cyclic AMP was somewhat slower in onset (peak at 15 min of incubation) than the G12a phosphorylation induced by the protein kinase C activators (peak at 5 min). This may reflect ( u ) the time taken for permeation of this lipid-soluble cyclic AMP analogue to its site of action or (6) an indirect mode of action. The latter explanation is supported by the finding that G12a lacks a known consensus sequence for A-kinase-mediated phosphorylation [ 11. Phosphorylation of G12a was associated with loss of G, function as assessed by the ability of low concentrations ( 1 L