Figure 4. Role of G-proteins in signaling of recombinantly expressed olfactory receptors.

(A) Chimeric construct with N-terminus of human Gα₁₆ and the C-terminus of Drosophila Gα-proteins. (B) Ratio of transfected HEK293 cells responding to 500 µM cyclohexanone in Ca²⁺ imaging experiments; cells either express OR43a, OR83b and the respective G-protein chimera, OR43a alone and the respective G-protein chimera, or OR43a, OR83b and full length human Gα₁₆. An increase in the ratio means that more cells responded upon co-expression of the G-protein chimera (n>5 independent transfections). The ratio determined with the Gα_s chimera was compared to the ratios obtained with the other G-protein chimera and was found to be signify-cantly different from all of them. (C) Western blot detection of recombinantly expressed G-protein chimera with an antibody against Gα₁₆, equal amounts of protein was loaded per lane (controlled by GAPDH detection), control (ctrl) were non-transfected HEK293 cells. (D) Quantification of western blot analysis by densiometry, G-protein and GAPDH bands were ana-lyzed from cell preparations from 3 independent transfections. Band intensities originating from Gα₁₆ stained membranes were divided by band intensities from GAPDH stained membranes. The small differences between the intensities from the different chimera were not significant (tested with Student's t-test). (E) [³⁵S]GTPγS binding to membrane preparations upon odorant stimu-lation, cells were transfected with ORs and Gα-protein, control cells were mock transfected. The reaction was carried out in triplicates. Differences between the indicated data points were statistically checked by the unpaired Student's t test, **p<0.01. Error bars represent s.e.m.