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

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)alpha. 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 alpha 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)alpha fusion protein, and both constructs were shown to interact with and activate endogenously expressed G(s)alpha. 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)alpha fusion. However, the fusion proteins containing either G(s)alpha or G(i1)-G(s)6alpha 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)6alpha 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)alpha-containing fusion protein and a 9-fold improvement when using the fusion protein containing G(i1)-G(s)6alpha to detect G-protein activation compared with expression of the isolated receptor.