Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins

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_sα. 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_s6 (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 [³H]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_sα fusion protein, and both constructs were shown to interact with and activate endogenously expressed G_sα. 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_i1α fusion. However, the fusion proteins containing either G_sα or G_i1-G_s6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G_i1-G_s6α 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_sα-containing fusion protein and a 9-fold improvement when using the fusion protein containing G_i1-G_s6α to detect G-protein activation compared with expression of the isolated receptor.