RGD Reference Report - Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.

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Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.
Authors:	Sakihama, T Masuda, K Sato, T Doi, T Kodama, T Hamakubo, T
Citation:	Sakihama T, etal., J Biotechnol. 2008 May 20;135(1):28-33. doi: 10.1016/j.jbiotec.2008.02.022. Epub 2008 Mar 7.
RGD ID:	8553387
Pubmed:	PMID:18403039 (View Abstract at PubMed)
DOI:	DOI:10.1016/j.jbiotec.2008.02.022 (Journal Full-text)
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.

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Object Symbol	Species	Term	Qualifier	Evidence	With	Notes	Source	Original Reference(s)
Gnas	Rat	adenylate cyclase-activating dopamine receptor signaling pathway	involved_in	IDA		PMID:18403039	BHF-UCL
Gnas	Rat	cellular response to catecholamine stimulus	involved_in	IDA		PMID:18403039	BHF-UCL
Gnas	Rat	cellular response to prostaglandin E stimulus	involved_in	IDA		PMID:18403039	BHF-UCL

Cellular Component

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Object Symbol	Species	Term	Qualifier	Evidence	With	Notes	Source	Original Reference(s)
Gnas	Rat	heterotrimeric G-protein complex	part_of	IDA		PMID:18403039	BHF-UCL

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Genes (Rattus norvegicus)

Gnas (GNAS complex locus)

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Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.