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 membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity 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 thereby targets it to its receptor substrate. The amino terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G(beta gamma), binding sequences, the targeting domain. We prepared this domain as a recombinant His(6) fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G(beta gamma), activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. G(i alpha)-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this Hiss fusion protein 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.