Kd and Bmax values for [3H] 5-CT binding to membranes of Sf9 cells coexpressing the h5-ht5A receptor and G protein subunits of the Gi/o, Gs, Gq/11, and G12/13 family
| Proteins Expressed | No Addition | + 100 μM Gpp(NH)p | ||||
|---|---|---|---|---|---|---|
| Kd | Bmax | n | Kd | Bmax | n | |
| nM | pmol/mg protein | nM | pmol/mg protein | |||
| h5-ht5A-HEK 2931-a | 2.3 ± 0.71-c | 31 ± 41-c | 6 | 4.3 ± 0.9 | 29 ± 7 | 4 |
| High | 0.4 ± 0.2 | 8 ± 4 | ||||
| Low | 5.5 ± 2.0 | 25 ± 4 | ||||
| h5-ht5A-Sf9 | 7.8 ± 0.9 | 39 ± 12 | 3 | 7.1 ± 0.1 | 32 ± 8 | 3 |
| h5-ht5A + Gβ1γ2-Sf9 | 5.6 ± 1.61-d | 29 ± 9 | 3 | 8.7 ± 1.11-f | 32 ± 10 | 3 |
| h5-ht5A + Gαi/o-Sf91-b | 3.7 ± 0.81-d | 16 ± 1 | 3 | 5.4 ± 0.8 | 15 ± 3 | 2 |
| h5-ht5A + Gαi/o + Gβ1γ2-Sf91-b | 0.8 ± 0.21-e | 8 ± 2 | 4 | 6.9 ± 1.51-f | 5 ± 4 | 3 |
| h5-ht5A + Gαi1-Sf9 | 5.2 ± 1.71-d | 46 ± 9 | 5 | 4.6 ± 0.6 | 38 ± 7 | 3 |
| h5-ht5A + Gαi1 + Gβ1γ2-Sf9 | 1.7 ± 0.71-e | 19 ± 3 | 6 | 5.9 ± 2.01-f | 19 ± 2 | 3 |
| h5-ht5A + Gαi2-Sf9 | 4.2 ± 1.11-d | 29 ± 6 | 5 | 5.4 ± 1.9 | 31 ± 14 | 3 |
| h5-ht5A + Gαi2 + Gβ1γ2-Sf9 | 1.9 ± 0.51-e | 18 ± 3 | 6 | 7.5 ± 4.01-f | 19 ± 3 | 3 |
| h5-ht5A + Gαi3-Sf9 | 4.8 ± 1.01-d | 28 ± 8 | 5 | 5.1 ± 1.0 | 22 ± 2 | 3 |
| h5-ht5A + Gαi3 + Gβ1γ2-Sf9 | 1.8 ± 0.61-e | 25 ± 6 | 6 | 6.0 ± 1.21-f | 30 ± 8 | 3 |
| h5-ht5A + Gαo-Sf9 | 8.0 ± 1.9 | 29 ± 5 | 4 | 8.0 ± 0.7 | 30 ± 5 | 4 |
| h5-ht5A + Gαo + Gβ1γ2-Sf9 | 2.0 ± 0.31-e | 42 ± 6 | 4 | 6.8 ± 0.71-f | 47 ± 8 | 4 |
| h5-ht5A + Gαz-Sf9 | 8.1 ± 0.9 | 26 ± 5 | 3 | 6.8 ± 1.9 | 24 ± 1 | 2 |
| h5-ht5A + Gαz + Gβ1γ2-Sf9 | 7.7 ± 1.8 | 24 ± 3 | 3 | 7.3 ± 0.3 | 24 ± 1 | 2 |
| h5-ht5A + Gαs-s-Sf9 | 7.2 ± 3.1 | 45 ± 3 | 5 | 6.4 ± 3.3 | 47 ± 4 | 3 |
| h5-ht5A + Gαs-s + Gβ1γ2-Sf9 | 7.4 ± 2.1 | 34 ± 6 | 4 | 7.6 ± 1.9 | 30 ± 7 | 3 |
| h5-ht5A + Gαq-Sf9 | 5.6 ± 0.71-d | 67 ± 12 | 3 | 6.6 ± 0.4 | 68 ± 6 | 3 |
| h5-ht5A + Gαq + Gβ1γ2-Sf9 | 6.0 ± 0.9 | 51 ± 6 | 3 | 7.0 ± 1.8 | 59 ± 7 | 3 |
| h5-ht5A + Gα11-Sf9 | 6.6 ± 2.4 | 42 ± 9 | 4 | 6.5 ± 2.5 | 38 ± 9 | 4 |
| h5-ht5A + Gα11 + Gβ1γ2-Sf9 | 5.0 ± 0.8 | 38 ± 9 | 3 | 4.5 ± 1.2 | 35 ± 12 | 3 |
| h5-ht5A + Gα16-Sf9 | 7.1 ± 0.8 | 58 ± 5 | 3 | 7.1 ± 1.0 | 55 ± 10 | 3 |
| h5-ht5A + Gα16 + Gβ1γ4-Sf9 | 6.9 ± 1.8 | 57 ± 13 | 3 | 7.8 ± 1.0 | 60 ± 15 | 3 |
| h5-ht5A + Gα12-Sf9 | 7.7 ± 3.3 | 11 ± 1 | 4 | 8.9 ± 6.5 | 12 ± 3 | 3 |
| h5-ht5A + Gα12 + Gβ1γ2-Sf9 | 10.7 ± 2.0 | 9 ± 2 | 3 | 8.5 ± 4.1 | 7 ± 2 | 2 |
| h5-ht5A + Gα13-Sf9 | 6.6 ± 2.6 | 27 ± 9 | 4 | 6.5 ± 2.6 | 27 ± 6 | 4 |
| h5-ht5A + Gα13 + Gβ1γ2-Sf9 | 6.9 ± 1.6 | 24 ± 5 | 3 | 6.2 ± 1.6 | 23 ± 6 | 3 |
Radioligand binding studies were performed, and Kdand Bmax values were derived as described inExperimental Procedures. The results are mean ± S.D. values from n independent experiments.
↵1-a Data for h5-ht5A-HEK 293 were taken fromFrancken et al., 1998. High and low denote the Kdand Bmax values of high- and low-affinity agonist binding site, respectively.
↵1-b Gαi/o represents the combination of Gαi1, Gαi2, Gαi3, and Gαosubunits.
↵1-c Values obtained from curve-fitting to a one-binding-site model, although a significantly better fit was achieved with a two-binding-site model (F test, P < .005).
↵1-d Kd value (for h5-ht5A receptors coexpressed with a Gα subunit or with Gβ1γ2that is significantly (Student's t test, P< .05) different from the Kd value for h5-ht5A-Sf9.
↵1-e Kd value (for h5-ht5A receptors coexpressed with a Gα subunit plus Gβ1γ2 that is significantly (Student's t test, P < .05) different from the Kd value for h5-ht5A+ Gβ1γ2-Sf9.
↵1-f Kd value of [3H]5-CT binding in the presence of 100 μM Gpp(NH)p that is significantly (Student'st test, P < .05) different from theKd value in the absence of Gpp(NH)p.