Bd. Blackhart et al., THE ANION-BINDING EXOSITE IS CRITICAL FOR THE HIGH-AFFINITY BINDING OF THROMBIN TO THE HUMAN THROMBIN RECEPTOR, Growth factors, 11(1), 1994, pp. 17-28
The thrombin receptor has been shown to be a novel member of the famil
y of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaten, V
.I., and Coughlin, S.R. (1991) Cell 64, 1057-1068). This receptor appe
ars to be activated through a thrombin-mediated proteolytic mechanism
which exposes a ''tethered ligand'' responsible for receptor activatio
n. In order to investigate the initial interactions of thrombin with t
his receptor, we have constructed cell lines which express high levels
of the human thrombin receptor and studied the binding of various for
ms of thrombin to the cell surface. Analysis of transfected cells with
thrombin receptor monoclonal antibodies identified a particular cell
line (clone #5-18) which displayed > 150,000 thrombin receptors per ce
ll. Clone #5-18 appeared to express functional receptors since treatme
nt with thrombin resulted in both a 15-20 fold increase of cytoplasmic
phosphoinositide levels and a comparable shift in the EC(50) of throm
bin-mediated calcium mobilization when compared to non-transfected CHO
cells. Binding of I-125-alpha-thrombin to clone #5-18 did not reach e
quilibrium at 37 degrees C. However, direct binding studies of I-125-a
lpha-, I-125-diisopropylphospho (DIP)-alpha-, and I-125-beta-thrombin
to clone #5-18 demonstrated that binding at 4 degrees C was saturable
and reversible for each ligand. Analysis of the binding data revealed
K-d's of 0.8 nM, 0.7 nM and 9.7 nM for I-125-DIP-alpha- and I-125-beta
-thrombin respectively. Association of I-125-alpha-, DIP-alpha, and be
ta-thrombin could be competed by unlabelled alpha- and DIP-alpha-throm
bin. Unlabelled beta-thrombin, which has a modified anion-binding exos
ite, was a poor competitor for I-125-DIP-alpha-thrombin, but did compe
te for I-125-beta-thrombin.