Gs alpha mediates epidermal growth factor-elicited stimulation of rat cardiac adenylate cyclase.

B G Nair,B Parikh,G Milligan,T B Patel

doi:10.1016/s0021-9258(17)45362-2

B G Nair, B Parikh + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)45362-2

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Abstract

In an earlier study we demonstrated that epidermal growth factor (EGF) increases the cellular accumulation of cAMP in perfused rat hearts by stimulating the cardiac adenylate cyclase via a stimulatory GTP-binding protein (Nair, B. G., Rashed, H. M., and Patel, T. B. (1989) Biochem. J. 264, 563-571). Employing antiserum, CS1, generated against a synthetic decapeptide RMHLRQYELL representing the carboxyl terminus of Gs alpha, the involvement of Gs in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. The CS1 antiserum specifically recognized two forms, (52 and 40 kDa) of Gs alpha in rat cardiac membranes; the 52 kDa being the predominant species. In functional assays of adenylate cyclase activity, the CS1 antiserum did not alter either aluminum fluoride- or forskolin-stimulated adenylate cyclase activity. Similarly, basal adenylate cyclase activity in the absence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was also not altered by the CS1 antiserum. However, as compared with controls performed in the presence of non-immune serum, preincubation of cardiac membranes with the CS1 antiserum resulted in a concentration-dependent inhibition of Gpp(NH)p-, isoproterenol-, and EGF-stimulated activities. In experiments which monitored Gi function as the ability of different G(pp)NHp, (-)N6-(R-phenylisopropyl)adenosine and carbachol to inhibit forskolin-stimulated adenylate cyclase, CS1 antiserum by inhibiting Gs, increased the apparent activity of Gi. Overall, our data demonstrate that the CS1 antiserum can specifically inhibit Gs function and therefore the stimulation of adenylate cyclase by agonists whose actions are mediated by Gs. In this respect, the data presented here demonstrate that Gs is the G-protein involved in mediating EGF-elicited stimulation of cardiac adenylate cyclase. Additionally, the finding that CS1 antiserum can overcome the effects of Gpp(NH)p on Gs, but not Gi, suggests that the carboxyl-terminal region of Gs alpha is important in the interactions with GTP or its analogs.

Highlights

From the recent studies which have demonstrated that the protein tyrosine kinase activity of the Epidermal growth factor (EGF) receptor phosphorylates phospholipase C in A431 cells as well as purified phospholipase C-7, it would appear that activation of phospholipase C in A431 cells involves phosphorylation of the protein on tyrosine residue(s) (Wahl et al, 1988, 1989, 1990; Nishibe et al, 1989, Kim et al, 1990)
Our studies demonstrated that EGF stimulates cardiac adenylate cyclase activity via a G-protein, and based on our findings with pertussis toxin and cholera toxin we suggested that EGF-elicited stimulation of adenylate cyclase in the heart involved the stimulatory G-protein, G, (Nair et al, 1989)
The non-immune serum recognized a low molecular mass protein band of -25 kDa which was not identified by the CSl antiserum

Summary

RMHLRQYELL representing the carboxyl terminus of

G,, the involvement of G. in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. Included among these are stimulation of metabolic processes such as glycolysis (Schneider et al, 1978), glycogenolysis (Bosch et al, 1987; Johnson and Garrison, 1987), alterations in the rate of metabolite transport processes (Moule and McGivan, 1987), and modulation of ion fluxes across the plasma membrane (Macara, 1986) These actions of EGF are manifested following the activation of different second messenger systems. Our studies demonstrated that EGF stimulates cardiac adenylate cyclase activity via a G-protein, and based on our findings with pertussis toxin and cholera toxin we suggested that EGF-elicited stimulation of adenylate cyclase in the heart involved the stimulatory G-protein, G,. CSl, against the synthetic carboxyl-terminal decapeptide, RMHLRQYELL, of G., (Milligan and Unson, 1989; Milligan et al, 1989), in this paper, we have presented experimental evidence to directly demonstrate that EGF, like the P-adrenergic agonist isoproterenol, stimulates cardiac adenylate cyclase via G,, activation

AND METHODS

RESULTS AND DISCUSSION

TABLE I

No addition

TABLE II

Adenylate cyclase activity