Assessment of the activation state of wild-type and mutant _IIbβ₃ in transiently transfected 293 cells. (A) Wild-type _IIb cDNA was transfected with wild-type β₃ cDNA (WT) (a through c) or mutant β₃ cDNAs (T562N [d through f], T562A [g through i], T562Q [j through l]) to 293 cells, and PAC1 binding was determined. Plots in the upper row (a, d, g, and j) represent nonspecific PAC1 binding determined in the presence of FK633. Plots in the lower row (c, f, i, and l) represent maximal PAC1 binding in the presence of PT25-2. Plots in the middle row (b, e, h, and k) represent PAC1 binding in the absence of the antagonist and the activating antibody. (B) Wild-type _IIbβ₃, _IIbβ₃(T564A), or _IIbβ₃(T562N,T564A) transfected cells were incubated with PAC1 and biotinylated-AP3, a non-function blocking anti-β₃ antibody, followed by incubation with FITC-conjugated antimouse IgM and PE-conjugated streptoavidin, and analyzed by flow cytometry. The overlay histogram represents PAC1 binding to cells expressing high levels of _IIbβ₃ determined by AP3 (denoted by the rectangle in the dot blots) [wild-type _IIbβ₃, dotted line; _IIbβ₃(T564A), solid line; _IIbβ₃(T562N,T564A), bold line]. (C) Wild-type and mutant _IIbβ₃ were surface-labeled with biotin, and immunoprecipitation was performed with AP2. Immnoprecipitates were electrophoresed on 6% polyacrylamide gel under reducing conditions. After transfer, membrane was incubated with peroxidase-conjugated avidin and developed with chemiluminescence.