Agonist Regulation of Cellular Gs[alpha] Levels in Wild-Type and Transfected NG108-15 Cells

The neuroblastoma x glioma hybrid cell line, NG108-15, expresses an apparent 1 - 1.5 pmoles of a high affinity IP prostanoid receptor. This receptor is coupled to Gs and activates adenylyl cyclase. Chronic exposure of these cells to the prostanoid agonist, iloprost, results in a desensitisation of prostanoid stimulated adenylyl cyclase activity. In agreement with earlier work (Kelly et al, 1990; McKenzie and Milligan, 1990), studies presented in this thesis demonstrate a 50-70% decrease in cellular Gsalpha levels on prostanoid treatment of NG108-15 cells. Time courses and dose responses of prostanoid treatment showed the down-regulation of Gsalpha to be concurrent with a down-regulation (50-70%) of the IP prostanoid receptor. Quantitative analysis of the polypeptide levels, on iloprost treatment, revealed that Gsalpha and the IP prostanoid receptor were down-regulated in a ratio of approximately 8:1 at various degrees of receptor occupancy by agonist. Although G-protein alpha-subunit down-regulation is now a well-documented phenomenon during long-term agonist exposure, it does not occur in all cases of chronic agonist treatment. In NG108-15 cells chronic agonist treatment of the A2 adenosine and secretin receptors, which also couple to adenylyl cyclase, does not result in a detectable down-regulation of Gsalpha (McKenzie et al, 1990). These receptors are thought to be expressed at much lower levels than the IP prostanoid receptor (Kelly et al, 1990; Gossen et al, 1990) potentially activating less G-protein alpha-subunit in NG108-15 cells. NG108-15 expressing 4000 fmoles/mg membrane protein beta2-adrenergic receptor (betaN22 cells) demonstrate an approximate 50% down-regulation of Gsalpha on treatment with isoprenaline (10muM; 16 hours), whereas NG 108-15 cells expressing 300 fmoles/mg membrane protein beta2-adrenergic receptor (betaN17 cells) show no apparent down-regulation of Gsalpha on prolonged isoprenaline treatment. These results show that isoprenaline mediated down-regulation of Gsalpha in transfected NG108-15 cells is dependent on the level of beta2-adrenergic receptor expression, indicating a role for the receptor in regulating Galpha levels. This could potentially prove to be a general phenomenon for other receptors and their related G-proteins