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(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.
Fong
C W
CW
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Milligan
G
G
D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D018076:DNA, Complementary; C113936:PTGIR protein, human; C491909:Ptgir protein, rat; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D002772:Cholera Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D016285:Iloprost