High Affinity Binding of β-Adrenergic Receptor Kinase to Microsomal Membranes

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The β-adrenergic receptor kinase (βARK) modulates β-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that βARK 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 5or, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of βARK. In this report, we used recombinant [35S]methionine-labeled βARK 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 βARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound βARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AlF4− or mastoparan/guanosine 5′-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated βARK. Interestingly, βARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of βARK1 (the proposed location of the βγ 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 ∼60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between βARK 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 βARK. The β-adrenergic receptor kinase (βARK) modulates β-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that βARK 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 5or, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of βARK. In this report, we used recombinant [35S]methionine-labeled βARK 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 βARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound βARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AlF4− or mastoparan/guanosine 5′-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated βARK. Interestingly, βARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of βARK1 (the proposed location of the βγ 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 ∼60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between βARK 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 βARK.