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High affinity binding of beta-adrenergic receptor kinase to microsomal membranes. Modulation of the activity of bound kinase by heterotrimeric G protein activation.

The beta-adrenergic receptor kinase (beta ARK) modulates beta-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that beta ARK also associates with intracellular microsomal membranes both "in vitro" and "in situ" (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and Mayor, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of beta ARK. In this report, we used recombinant [35S]methionine-labeled beta ARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 +/- 1 nM) present in salt-stripped rat liver microsomal membranes. Although beta ARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound beta ARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AIF4- or mastoparan/guanosine 5'-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated beta ARK. Interestingly, beta ARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of beta ARK1 (the proposed location of the beta gamma subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an approximately 60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between beta ARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of beta ARK.