Selective Activation of a Chimeric Gi1/GsG Protein α Subunit by the Human IP Prostanoid Receptor: Analysis Using Agonist Stimulation of High Affinity GTPase Activity and [35S]Guanosine-5′-O-(3-thio)triphosphate Binding
- 1Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland (C.W.F., D.S.B., G.M.) and 2Receptor Systems Unit, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, SG1 2NY, England (S.R.)
Abstract
A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [35S]guanosine-5′-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either Gsα or Gi1α. In contrast, coexpression of a chimeric G protein α subunit in which the carboxyl-terminal six amino acids of Gi1α were altered to those of Gsα resulted in robust stimulation by iloprost. Because the chimeric G protein α subunit (Gi1/Gs6α) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and Gi1/Gs6α with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as Gi1/Gs6α that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the Gi family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.
Footnotes
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Send reprint requests to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland. E-mail: g.milligan{at}bio.gla.ac.uk
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This work was supported by the Medical Research Council and the Biotechnology and Biosciences Research Council (UK). C.W.F. received a studentship from the National Science and Technology Board of Singapore. D.S.B. received a CASE studentship from the Biotechnology and Biosciences Research Council.
- Abbreviations:
- FhIPR
- FLAG-tagged human IP prostanoid receptor
- hIPR
- human IP prostanoid receptor
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- PCR
- polymerase chain reaction
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HEK
- human embryonic kidney
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- PBS
- phosphate-buffered saline
- GPCR
- G protein-coupled receptor
- DOTAP
- N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
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- Received February 24, 1998.
- Accepted April 14, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
