JANUS KINASE-2 (JAK2) MUST BE CATALYTICALLY ACTIVE TO ASSOCIATE WITH THE AT(1) RECEPTOR IN RESPONSE TO ANGIOTENSIN-II

Angiotensin II evokes a variety of biological responses by binding to a seven transmembrane cell surface receptor termed AT,. Ligand binding to the AT(1) receptor induces the physical association and activation of the intracellular kinase Jak2. To elucidate the mechanism of this association, COS-7 cells were cotransfected with the AT, receptor and either wild type Jak2 or a catalytically inactive Jak2. AT, receptor-J ak2 association was assessed in vitro by a GST-AT(1) receptor fusion p rotein binding assay and in vivo by direct co-immunoprecipitation of t he receptor-Jak2 complex. Both studies showed that Jak2 must be cataly tically active to form a complex with the AT1 receptor, and that compl ex formation is associated with Jak2 tyrosine phosphorylation. These r esults were confirmed using the Jak2 specific inhibitor AG-490. We als o found that over-expression of wild type Jak2 in COS-7 cells leads to in vivo complex formation of spontaneously autophosphorylated Jak2 wi th the AT, receptor. No such complex formation was observed with a dom inant negative Jak2. Thus, the physical association of Jak2 with the A T1 receptor is regulated by an angiotensin II mediated autophosphoryla tion event. (C) 1998 Academic Press.