Association of the regulatory beta-adrenergic receptor kinase with rat liver microsomal membranes.
beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.
García-Higuera
I
I
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Penela
P
P
Murga
C
C
Egea
G
G
Bonay
P
P
Benovic
J L
JL
Mayor
F
F
D000818:Animals; D000831:Animals, Newborn; D002451:Cell Compartmentation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D012243:Rhodopsin / Q000378:metabolism; D014018:Tissue Distribution; D051552:beta-Adrenergic Receptor Kinases
D012243:Rhodopsin; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases