Table 1.
Summary of ligand binding properties and expressions of the wild-type TP, IP, and chimeric receptors
| Receptor | Intracellular loop(s) | Kda,b (nM) | Bmaxa,c (pmol/mg) | Cell surfaced expression (%) | EC50e Ca2+ (nM) | EC50f cAMP (nM) |
|---|---|---|---|---|---|---|
| Wild-type TP | 5.8 ± 1.3 | 5.1 ± 0.3 | 100 | 13.1 ± 0.8 | ND | |
| Wild-type IP | 108 ± 1 | ND | 7.7 ± 0.3 | |||
| TP ICL1-IP | ICL1 | 4.8 ± 1.1 | 8.9 ± 0.4 | 96 ± 7 | 4.6 ± 1.1 | ND |
| TP ICL2-IPg | ICL2 | ND | 93 ± 7 | 8.3 ± 0.5 | 22.7 ± 0.3 | |
| TP ICL2A-IP | ICL2 | 4.3 ± 0.6 | 3.1 ± 0.3 | 76 ± 6 | 11.0 ± 0.4 | 51.2 ± 1.3 |
| TP ICL2B-IP | ICL2 | 5.3 ± 0.8 | 9.3 ± 0.4 | 108 ± 7 | 15.6 ± 2.0 | 62.2 ± 1.2 |
| TP ICL3-IPg | ICL3 | ND | 42 ± 4 | |||
| TP ICL3A-IP | ICL3 | ND | 49 ± 7 | |||
| TP ICL3B-IPg | ICL3 | ND | 92 ± 2 | 32.4 ± 1.3 | 39.8 ± 1.1 | |
| TP ICL3C-IP | ICL3 | 5.8 ± 1.2 | 3.4 ± 0.4 | 89 ± 1 | 61.6 ± 2.1 | ND |
| TP ICL2B-ICL3B–IP | ICL2/3 | 8.1 ± 3.9 | 0.9 ± 0.2 | 97 ± 2 | 30.7 ± 0.2 | 18.3 ± 0.2 |
| TP ICL2B-ΔYLYAQh | ICL2 | 7.7 ± 4.0 | 0.7 ± 0.2 | 81 ± 9 | 37.5 ± 0.2 | ND |
| R60L | ICL1 | 9.8 ± 1.3 | 1.3 ± 0.2 | 85 ± 2 | 44.9 ± 0.4 | |
| TP ICL2B-R60L | ICL1/2 | 19.7 ± 2.0 | 2.5 ± 0.2 | 96 ± 2 | 12.1 ± 0.4 | |
| TP ICL2B-3B–IP–R60L | ICL1/2/3 | 21.7 ± 3.9 | 4.1 ± 0.2 | 92 ± 1 | 17.0 ± 0.2 | |
| T135A | ICL2 | 7.6 ± 2.6 | 5.7 ± 0.8 | 72 ± 12 | ND | |
| R136A | ICL2 | 8.7 ± 1.9 | 5.5 ± 0.5 | 81 ± 17 | 14.2 ± 0.4 | |
| R147A | ICL2 | 9.2 ± 2.3 | 6.0 ± 0.6 | 82 ± 17 | ND | |
| R148A | ICL2 | 7.3 ± 2.1 | 9.8 ± 0.1 | 46 ± 12 | 12.2 ± 0.3 | |
| H224Ag | ICL3 | ND | 49 ± 10 | |||
| V225Ag | ICL3 | ND | 88 ± 8 | |||
| E230Ag | ICL3 | ND | 72 ± 2 | 8.0 ± 0.2 | ||
| Q252Ag | ICL3 | ND | 97 ± 19 | 2.1 ± 0.2 |
The values are expressed as the mean ± standard error (SE) of 3 to 5 experiments in duplicate performed using the TP antagonist 3H-labeled SQ 29548 as the radioligand (product no. NET936250UC, PerkinElmer). ND, not determined.
Kd, affinity of the antagonist SQ 29548 for the receptor.
Bmax, binding maximum of the ligand SQ 29548 for the receptor, expressed as pmol of the TP receptor per mg of total membrane protein.
Cell surface expression of the receptor determined using flow cytometry (see Materials and Methods), represented using the wild-type TP set at 100%.
EC50s in this column indicate the molar concentrations of the agonist U46619 that produce 50% of the maximal possible effect (calcium mobilization) for the TP and chimeric or mutant receptors. A one-way ANOVA of the EC50 values for calcium mobilized between the wild-type TP and the chimeric receptors and mutants showed a significant difference (P < 0.05).
EC50s in this column indicate the molar concentrations of the agonist iloprost for IP and of U46619 for the TP and chimeric or mutant receptors that produce 50% of the maximal possible effect (cAMP production).
No significant specific binding to the antagonist 3H-labeled SQ 29548 and/or dose-dependent response to the agonist for these receptors was detected under our assay conditions.
TP ICL2B-IP with the YLYAQ sequence in the middle of ICL2B removed.