Stimulation of increases in intracellular calcium and prostaglandin E2 generation in Chinese hamster ovary cells expressing receptor-Galpha16 fusion proteins.
We examined whether fusion proteins of G protein-coupled receptors with the alpha subunit of G(16) (Galpha(16)) could activate downstream signals. We expressed fusion proteins of G(i)-coupled receptors, i.e. CX(3)C chemokine receptor 1 (CX(3)CR1) and M(2) receptor, in Chinese hamster ovary cells. An agonist for CX(3)CR1 induced greater increases in intracellular Ca(2+) and prostaglandin E(2) generation in cells expressing CX(3)CR1-Galpha(16) fusion protein than in cells expressing CX(3)CR1 alone or both CX(3)CR1 and Galpha(16) separately. Similarly, agonist-induced prostaglandin E(2) generation was greater in cells expressing M(2)-Galpha(16) fusion protein than ones expressing M(2) alone or both M(2) and Galpha(16) separately. In cells expressing fusion proteins with Galpha(16) of G(q)-coupled receptors, i.e. urotensin II receptor and M(1) receptor, the relevant agonists induced similar increases in intracellular Ca(2+) and prostaglandin E(2) generation as in ones expressing the receptor alone. In cells expressing urotensin II receptor-Galpha(16) fusion protein, prostaglandin E(2) generation exhibited a lower EC(50) value than the intracellular Ca(2+) increase. These results indicate that agonist-stimulated receptor-Galpha(16) fusion proteins are coupled to downstream signaling pathways, and suggest that receptor-Galpha(16) fusion proteins may be useful for screening for ligands of orphan G protein-coupled receptors and G(i)-coupled receptors.
Suga
Hinako
H
Department of Neurochemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033. hsuga@showa.gunma-u.ac.jp
Takeda
Shigeki
S
Haga
Tatsuya
T
Okamura
Michiko
M
Takao
Kyoichi
K
Tatemoto
Kazuhiko
K
D000818:Animals; D016466:CHO Cells; D000073883:CX3C Chemokine Receptor 1; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism; D011485:Protein Binding; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D019707:Receptors, Chemokine / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D011453:Prostaglandins; D043585:Receptor, Muscarinic M2; D019707:Receptors, Chemokine; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C121697:UTS2R protein, human; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D015232:Dinoprostone; D002118:Calcium