Table 4

Effects of replacement of amino acids in TM7 of the β2AR with alanine or phenylalanine on ligand-binding characteristics of the β2AR

125I-CYPLigands (Ki)
KdBmaxFormoterolProcaterolSalmeterol4-cIsoproterenol
pM pmol/mg nM
WT β2AR26.5  ± 7.02.1  ± 0.235  ± 8240  ± 267.4  ± 3.3190  ± 26
E306A-40.4  ± 8.40.2  ± 0.0428  ± 8140  ± 8
V307A-24.0  ± 2.02.6  ± 0.442  ± 0.3230  ± 34
Y308A-23.0  ± 2.05.5  ± 0.7740  ± 170b 2800  ± 340b 184  ± 60b 4-c 1300  ± 130b 4-c
I309A-27.1  ± 4.54.2  ± 0.7120  ± 28340  ± 82
L310A-26.9  ± 2.13.2  ± 0.226  ± 3150  ± 26
L311A-19.9  ± 3.23.6  ± 0.239  ± 4500  ± 120
I314A-23.7  ± 2.62.5  ± 0.323  ± 8150  ± 26
V317A-23.4  ± 6.13.1  ± 0.843  ± 1220  ± 19
G320A-33.0  ± 11.37.4  ± 1.446  ± 5300  ± 23
L324A-30.1  ± 11.33.5  ± 0.998  ± 10840  ± 614-a
Y308F-16.5  ± 1.34.4  ± 0.296  ± 171400  ± 300b 32  ± 3370  ± 98

The binding of ligands to the WT β2AR and alanine-substituted β2AR mutants were determined by competition with 50pM125I-CYP. The data were analyzed using the nonlinear least-squares regression computer program as described underExperimental Procedures. The results are shown as the mean ± S.E. from three to four separate experiments.

    • 4-a P < 0.05;bP < 0.01 compared with the WT β2AR.

    • 4-c The affinities of salmeterol or isoproterenol for the Y308A-β2AR were obtained from Isogaya et al. (1998) orKikkawa et al. (1998).