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
- Received February 24, 1998.
- Accepted April 14, 1998.
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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.
- The American Society for Pharmacology and Experimental Therapeutics
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