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 Gαq than by Gαi2, the high-affinity agonist binding to the 5-HT 1A receptor was preferentially enhanced by Gαi2 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 Gαi2 in preference to Gαq. In contrast, in situ reconstitution data show that 5-HT2C receptors robustly activate Gαq and marginally activate Gαo or Gαi, whereas 5-HT1A receptors only marginally activate Gαq and robustly activate Gαo and Gαi. 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.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- History and Philosophy of Science