TABLE 1

Effect of mutation of Arg188, Asn229, and Gln380 in human VPAC1

pIC50 of binding, pEC50 and Emax of adenylate cyclase activation for VIP on membranes from CHO cells expressing the wild-type VPAC1 or mutated receptor. ΔpEC50 is defined as pEC50(mutant) − pEC50(wt) and ΔpIC50 as pIC50(mutant) − pIC50(wt). The expected values are the changes that would be expected if there was strict additivity. Emax is measured in response to 1 μM VIP, and values represent the mean ± S.E.M. of at least three independent experiments.

Binding StudiesAdenylate Cyclase Assay
pIC50ΔpIC50Expected ΔpIC50Receptor DensitypEC50ΔpEC50Expected ΔpIC50Emax
pmol/mg proteinpmol/mg protein/min
Single mutants
    VPAC18.53 ± 0.072.1 ± 0.28.59 ± 0.09160 ± 3
    R188AaN.D.
    R188N7.08 ± 0.07*−1.452.0 ± 0.37.63 ± 0.11*−0.96140 ± 5
    R188Qa6.52 ± 0.05*−2.011.9 ± 0.27.72 ± 0.06*−0.87152 ± 3
    N229Ab8.42 ± 0.08−0.112.3 ± 0.47.36 ± 0.11*−1.2350 ± 4*
    N229Qb8.57 ± 0.100.042.2 ± 0.37.58 ± 0.16*−1.01111 ± 9*
    N229R8.65 ± 0.060.122.0 ± 0.38.48 ± 0.12−0.11123 ± 3*
    Q380A8.61 ± 0.080.081.9 ± 0.27.30 ± 0.10*−1.2951 ± 4*
    Q380N8.34 ± 0.08−0.192.3 ± 0.38.35 ± 0.09−0.20106 ± 3*
    Q380R7.18 ± 0.09*−1.352.4 ± 0.27.81 ± 0.12*−0.7843 ± 3*
Double mutants
    VPAC18.53 ± 0.072.1 ± 0.28.59 ± 0.09160 ± 3
    N229A/Q380A7.61 ± 0.09*−0.92−0.032.0 ± 0.37.14 ± 0.13*−1.45−2.5235 ± 5*
    R188N/N229RN.D.−1.336.61 ± 0.12*−1.98−1.07120 ± 4*
    R188Q/Q380R6.46 ± 0.04*−2.07−3.361.8 ± 0.36.41 ± 0.12*−2.18−1.6535 ± 2*
    N229Q/Q380N7.87 ± 0.08*−0.66−0.152.3 ± 0.28.08 ± 0.11−0.51−1.21117 ± 3*
  • N.D., not detectable.

  • * P < 0.05 evaluated by Mann-Whitney test.

  • a From Solano et al. (2001).

  • b From Nachtergael et al. (2006).