In vivo reconstitution of dopamine D2S receptor-mediated G protein activation in baculovirus-infected insect cells: preferred coupling to Gi1 versus Gi2

Biochemistry
S GrünewaldH Michel

Abstract

Agonist binding of the human D2S receptor overexpressed in baculovirus-infected Sf9 insect cells was of low affinity and GppNHp-insensitive, yet, dopaminergic agonists were able to partly inhibit forskolin-stimulated cAMP accumulation. In order to prove full functionality of the receptor, we used an "in vivo" reconstitution system, which is based on coinfection of Sf9 cells with the appropriate receptor and G protein encoding baculoviruses. In cells coexpressing the D2S receptor and either Gi1 or Gi2, the dopaminergic agonist apomorphine effectively stimulated [35S]GTP gamma S binding and GTPase activity. Agonist-stimulated [35S]GTP gamma S binding was dependent on the ratio of G protein to receptor. Expression levels of receptor and G protein influenced each other reciprocally. G protein activation could be optimized by varying the multiplicity of infection of the receptor and G protein encoding baculoviruses. Coexpression of either Gi1 or Gi2 led to the appearance of GppNHp-sensitive high-affinity agonist binding. Detailed agonist competition binding analysis revealed that the percentage of high-affinity agonist binding sites was significantly higher in D2S receptor-expressing cells coinfected with Gi1 viruses than when coinf...Continue Reading

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May 23, 2012·Molecular Neurobiology·Jing WuLing-Qiang Zhu
Sep 7, 2001·Biochemical Pharmacology·J NasmanK E Akerman
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