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 exchang e and hydrolysis are frequently impossible to monitor for receptors which i nteract predominantly with G(s)alpha. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp -Asp-Asp-Lys)-epitepe-tagged human IP prostanoid receptor and fusion protei ns generated between this form of the receptor and the alpha subunits of it s cognate G-protein G(s), G(il), a G-protein which it fails to activate in co-expression studies, and a chimaeric G(il)-G(s)6 (a form of G(il) in whic h 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 [H- 3]ligand-binding studies and by immunoblotting with both an anti-FLAG antib ody and with appropriate antisera to the G-proteins. Each construct display ed similar affinity to bind the agonist iloprost. Iloprost stimulated adeny late cyclase activity in clones expressing both IP prostanoid receptor and the IP proststnoid receptor-G(s)alpha fusion protein, and both constructs w ere shown to interact with and activate endogenously expressed G(s)alpha. A ddition 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-Gi,a fusion. However, the fusion proteins containin g either G(s)alpha or G(il)-G(s)6 alpha produced substantially greater stim ulation of GTPase activity than the isolated IP prostanoid receptor. Treatm ent of cells expressing the IP prostanoid receptor-G(il)-G(s)6 alpha fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normali zation of such results for levels of expression of the IP prostanoid recept or constructs demonstrated a 5-fold higher stimulation of GTPase activity w hen using the G(s)alpha-containing fusion protein and a 9-fold improvement when using the fusion protein containing G(il)-G(s)6 alpha to detect G-prot ein activation compared with expression of the isolated receptor.