Comparison of G-protein selectivity of human 5-HT2C and 5-HT1A receptors.
We compared the ability of human 5-HT2C and 5-HT1A receptors to couple to selected G proteins expressed in insect Sf9 cells through simultaneous infection with recombinant baculoviruses. We also examined the coupling of G proteins to these same receptors in membranes derived from the Sf9 cells using in situ reconstitution with purified G proteins. Our data show that unoccupied 5-HT2C and 5-HT1A receptors can attain an activated conformation that is stabilized by interaction with specific G proteins. While high-affinity agonist binding to the 5-HT2C receptor was increased to a greater extent by Galphaq than by Galphai2, the high-affinity agonist binding to the 5-HT1A receptor was preferentially enhanced by Galphai2 coexpression. When the two 5-HT receptors were expressed in cells also expressing G proteins, both 5-HT2C and 5-HT1A receptors appear to activate Galphai2 in preference to Galphaq. In contrast, in situ reconstitution data show that 5-HT2C receptors robustly activate Galphaq and marginally activate Galphao or Galphai, whereas 5-HT1A receptors only marginally activate Galphaq and robustly activate Galphao and Galphai. These results suggest that the overexpression of receptor and potential G-protein coupling partners in Sf9 cells may lead to erroneous conclusions as to the signaling selectivity of receptors.
Okada
Mitsuko
M
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852, USA. mokada@med.osaka-cu.ac.jp
Goldman
David
D
Linnoila
Markku
M
Iwata
Nakao
N
Ozaki
Norio
N
Northup
John K
JK
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004873:Ergolines / Q000378:metabolism / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007313:Insecta; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D058829:Serotonin 5-HT1 Receptor Antagonists; D058830:Serotonin 5-HT2 Receptor Antagonists
D004873:Ergolines; D044405:Receptor, Serotonin, 5-HT2C; D043562:Receptors, G-Protein-Coupled; D058829:Serotonin 5-HT1 Receptor Antagonists; D058830:Serotonin 5-HT2 Receptor Antagonists; D044282:Receptor, Serotonin, 5-HT1A; D020962:Heterotrimeric GTP-Binding Proteins; C031649:mesulergine