Signaling activities of wild-type and mutant cytokine receptors.

(A) Structures of wild-type and mutant receptors are diagrammed. Positions of the transmembrane domain (TM) and phosphorylatable tyrosine (Y) are indicated. (B) 32D cells stably expressing wild-type (wt) or mutated EPO-R were left unstimulated (−) or were stimulated with 1.4 ng/mL IL-3 or 5 U/mL EPO for 8 minutes. Akt catalytic activity was assayed from cell lysates in an in vitro kinase assay (see “Materials and methods”). Alternatively, whole cell lysates were Western blotted with anti–phospho-Erk (α P-Erk) or anti–phospho-Stat-5A/B (α P-STAT5) antibodies. As a control for loading, lysates were probed with anti-Erk2 (α Erk) antiserum.