FUNCTIONALLY ACTIVE TARGETING DOMAIN OF THE BETA-ADRENERGIC-RECEPTOR KINASE - AN INHIBITOR OF G-BETA-GAMMA-MEDIATED STIMULATION OF TYPE-II ADENYLYL-CYCLASE

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its memb rane-associated receptor substrates, such as the beta-adrenergic recep tor, triggering events leading to receptor desensitization. beta ARK a ctivity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G(beta gamma)), which translocates the kinase to the plasma membrane and the reby targets it to its receptor substrate. The amino terminal two-thir ds of beta ARK1 composes the receptor recognition and catalytic domain s, while the carboxyl third contains the G(beta gamma), binding sequen ces, the targeting domain. We prepared this domain as a recombinant Hi s(6) fusion protein from Escherichia coli and found that it had both i ndependent secondary structure and functional activity. We demonstrate d the inhibitory properties of this domain against G(beta gamma), acti vation of type II adenylyl cyclase both in a reconstituted system util izing Sf9 insect cell membranes and in a permeabilized 293 human embry onic kidney cell system. G(i alpha)-mediated inhibition of adenylyl cy clase was not affected. These data suggest that this Hiss fusion prote in derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G(beta gamma)-mediated processes and for studying the structural features of a G(beta gamma)-binding domain.