Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with Gsα. 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 Gs, Gi1, a G-protein which it fails to activate in co-expression studies, and a chimaeric Gi1-Gs6 (a form of Gi1 in which the C-terminal six amino acids were replaced with the equivalent sequence of Gs) 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-Gsα fusion protein, and both constructs were shown to interact with and activate endogenously expressed Gsα. 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-Gi1α fusion. However, the fusion proteins containing either Gsα or Gi1-Gs6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-Gi1-Gs6α 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 Gsα-containing fusion protein and a 9-fold improvement when using the fusion protein containing Gi1-Gs6α to detect G-protein activation compared with expression of the isolated receptor.
Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins
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Chee Wai FONG, Graeme MILLIGAN; Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins. Biochem J 1 September 1999; 342 (2): 457–463. doi: https://doi.org/10.1042/bj3420457
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