Construct | EC50 | |
---|---|---|
Thrombin | SFLLRNP-NH2 | |
nM | μM | |
Wild-type | 0.05 ± 0.04 | 0.5 ± 0.3 |
Deletions | ||
ΔNH3 (68–93) | 1 ± 0.8 | >10 |
ΔEC1 (163–174) | >10 | NT |
ΔEC2 (240–267) | >10 | NT |
ΔEC3 (339–344) | 0.1 ± 0.06 | >50 |
Point mutants | ||
F87A | 0.05 ± 0.04 | 0.5 ± 0.3 |
I88A | 0.05 ± 0.03 | 30 ± 15 |
S89A | 0.05 ± 0.04 | 30 ± 18 |
D91N | 0.05 ± 0.03 | 0.5 ± 0.2 |
S93A | 0.05 ± 0.02 | 0.5 ± 0.3 |
Y95A | 0.05 ± 0.04 | 0.5 ± 0.3 |
L96A | 0.05 ± 0.04 | 20 ± 10 |
EC1DE (D167A/E173A) | 0.05 ± 0.03 | 0.5 ± 0.3 |
EC2DE (E241A, D256A, E260A, E264A) | >10 | >40 |
E241A | 0.05 ± 0.04 | 0.5 ± 0.3 |
D256A | 0.1 ± 0.07 | >40 |
D256N | 0.05 ± 0.04 | 5 ± 3 |
E260A | 0.05 ± 0.03 | 0.5 ± 0.4 |
E264A | 0.05 ± 0.04 | 0.5 ± 0.3 |
E347A | 0.1 ± 0.08 | >50 |
E347Q | 0.5 ± 0.3 | >40 |
C175S | 10 ± 2 | >40 |
C175S, C254S | 10 ± 1 | >40 |
Wild-type or mutant PAR-1 receptors were expressed in X. laevis oocytes and EC50 values for 45Ca efflux in response to thrombin and SFLLRNP were evaluated. Responses were determined as a percentage of the control value, which was the maximum response of PAR-1 to either thrombin or SFLLRNP. EC50 values shown are representative of those obtained in at least two separate experiments performed in triplicate.
NT, not tested: the mutants that were not responsive to thrombin were not tested for TRAP responses.