Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.
Abstract
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 His6 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. Gi alpha-mediated inhibition of adenylyl cyclase
was not affected. These data suggest that this His6 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.
Type
Journal articleSubject
Adenylate Cyclase ToxinAdenylyl Cyclases
Amino Acid Sequence
Animals
Binding Sites
Circular Dichroism
Cyclic AMP-Dependent Protein Kinase Type II
Cyclic AMP-Dependent Protein Kinases
Enzyme Activation
GTP-Binding Proteins
Humans
In Vitro Techniques
Molecular Sequence Data
Protein Structure, Secondary
Rats
Receptors, Adrenergic, beta
Recombinant Fusion Proteins
Signal Transduction
Virulence Factors, Bordetella
beta-Adrenergic Receptor Kinases
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Robert J. Lefkowitz
The Chancellor's Distinguished Professor of Medicine
Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his
early career as a cardiologist and his transition to biochemistry, which led to his
Nobel Prize win.
Robert J. Lefkowitz, M.D. is James B. Duke Professor of Medicine and Professor of
Biochemistry and Chemistry at the Duke University Medical Center. He has been an Investigator
of the

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