Mutational Analysis of Gβγ and Phospholipid Interaction with G Protein-coupled Receptor Kinase 2

Abstract

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free G␤␥ subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with G␤␥ and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by G␤␥. Alternatively, mutation of residues distal to the C-terminal amphipathic ␣-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to G␤␥. Interestingly, mutation of Arg-587 in ␤-sheet 3, a region not previously thought to interact with G␤␥, resulted in a specific and profound loss of G␤␥ responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound G␤␥ similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind G␤␥. In order to examine the role of phospholipid and G␤␥ interaction in cells, wild-type and mutant GRK2s were expressed with a ␤ 2 -adrenergic receptor (␤ 2 AR) mutant that is responsive to GRK2 phosphorylation (␤ 2 AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of ␤ 2 AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the -opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for G␤␥ and phospholipids, and (ii) demonstrated that G␤␥ binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.Diverse extracellular stimuli are perceived at the plasma membrane by G protein-coupled receptors (GPCRs).1 Agonistoccupied receptors promote the activation and dissociation of the heterotrimeric G protein ␣ and ␤␥ subunits, each of which goes on to regulate various effector molecules thereby producing a physiological response. This process is regulated in an agonist-dependent fashion by a family of G protein-coupled receptor kinases (GRKs), which phosphorylate activated GPCRs promoting binding of a second family of proteins, termed arrestins, which serve to uncouple the GPCR from further G protein activation (1, 2). Arrestin binding also promotes receptor internalization, which facilitates the processes of receptor resensitization and down-regulation (1).In general, all G...

Inositol Lipid Binding and Membrane Localization of Isolated Pleckstrin Homology (PH) Domains

“…In the few cases where their sites of interaction were mapped, the determinants to bind inositides and those with ␤␥ subunits did not completely overlap, the latter mostly being found toward the C-terminal end of the PH domain (56,57). Intriguingly, a recent detailed study (58) localized some key residues important for ␤␥ binding of GRK2 to the C-terminal helix of its PH domain. Therefore, ␤␥ subunits are possible candidates to interact with PLC␦ 1 PH to aid its membrane localization.…”

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi, GRK2, and Arrestin3

“…sibility of linking G i to the phosphorylation of activated GPR109A via protein kinase A, but our observation that the siRNA knockdown of GRK2 completely inhibited GPR109A internalization indirectly excludes a role for protein kinase A in the regulation of GPR109A internalization. Many studies suggest that GRK2 activity also requires the binding of the G␤␥ subunit (28,59), and the plasma membrane translocation of these kinases is regulated by their association with the G␤␥ subunit (29,60). It is then more likely that PTX pretreatment abolishes GPR109A internalization by inhibiting the association of G␤␥ subunit with GRK2.…”

“…All pictures and data shown are representative of at least three independent experiments. GRK2 to the membrane to promote receptor phosphorylation (28,29). Thus, we next investigated if the G␤␥ subunit regulates GPR109A internalization.…”

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

“…Fig. 4A shows that expression of a R587Q GRK2 mutant, which lacks G␤␥ binding (25), was completely inactive in suppressing D 2 DAR-mediated inhibition of cAMP accumulation. These results suggest that G␤␥ interactions are necessary for GRK2-mediated suppression of D 2 DAR signaling.…”