In vivo reconstitution of dopamine D2S receptor-mediated G protein activation in baculovirus-infected insect cells: preferred coupling to Gi1 versus Gi2.
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 coinfected with Gi2 viruses. Moreover, the coexpressed Gi proteins seemed to modulate the affinity of agonists for the high-affinity form of the receptor. In cells coexpressing Gi1, agonist high affinity was 2-4-fold higher than in cells coexpressing Gi2. Na+ increased the dissociation constant of apomorphine for the high-affinity site by 2-4-fold without affecting the percentage of high-affinity sites or the preference for Gi1. In some dopamine competition experiments with coinfected cells, displacement data were best fit assuming three noninteracting classes of sites in the absence and two independent classes of sites in the presence of GppNHp. Dopamine competition curves with cells highly overexpressing the D2S receptor or with membranes from such cells were best fit assuming two independent classes of sites which were insensitive to GppNHp and might reflect abnormal compartimentalization and/or different states of aggregation.
Grünewald
S
S
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Reiländer
H
H
Michel
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001058:Apomorphine / Q000378:metabolism; D016367:Baculoviridae; D001667:Binding, Competitive; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018491:Dopamine Agonists / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D013134:Spiperone / Q000031:analogs & derivatives / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism / Q000821:virology*
D018491:Dopamine Agonists; D017448:Receptors, Dopamine D2; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013134:Spiperone; D006160:Guanosine Triphosphate; C039167:3-N-methylspiperone; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D001058:Apomorphine