The RGS (regulator of G-protein signalling) proteins are GTPase-activating proteins for activated Gα subunits. We investigated the effects of protein kinase C (PKC) on RGS proteins in various T cell lines by treating them with PMA. mRNA levels of both RGS16 and tumour necrosis factor α (TNFα) were found to be up-regulated in CEM leukaemia cells in a PKC-dependent manner. Mezerein, a non-phorbol-ester activator of PKC, also elevated RGS16 and TNFα mRNA levels, while the specific PKC inhibitor Go6983 abrogated their expression. In view of the slower kinetics of PMA-induced RGS16 expression and the tight correlation between TNFα and RGS16 mRNA induction among the cell lines studied, we suggest that activation of PKC up-regulates RGS16 via TNFα. Indeed, addition of recombinant TNFα to CEM cells rapidly stimulated RGS16 mRNA expression independently of PKC. Furthermore, mobilization of calcium by A23187 and thapsigargin blocked the TNFα-mediated induction of RGS16, which was reversed by EGTA and by the immunosuppressants FK506 and cyclosporin A, suggesting that the calcineurin/NF-AT (nuclear factor of activated T cells) pathway may repress the up-regulation process. Our results demonstrate for the first time that activation of PKC induces RGS16 expression via TNFα in a calcium-sensitive manner, thereby implicating RGS16 in the regulation of T cell responses to inflammation.
Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G s α. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein G s , G i1 , a G-protein which it fails to activate in co-expression studies, and a chimaeric G i1 -G s 6 (a form of G i1 in which the C-terminal six amino acids were replaced with the equivalent sequence of G s ) were stably expressed in HEK293 cells. These were detected by [ 3 H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-G s α fusion protein, and both constructs were shown to interact with and activate endogenously expressed G s α. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-G i1 α fusion. However, the fusion proteins containing either G s α or G i1 -G s 6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G i1 -G s 6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the G s α-containing fusion protein and a 9-fold improvement when using the fusion protein containing G i1 -G s 6α to detect G-protein activation compared with expression of the isolated receptor.