The β-Adrenergic Receptor Kinase (GRK2) Is Regulated by Phospholipids

Abstract

The ␤-adrenergic receptor kinase (␤ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). ␤ARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. ␤ARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein ␤␥ subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (␤ARK1) was found to require phospholipid in order to phosphorylate the ␤ 2 -adrenergic receptor. As determined with a nonreceptor peptide substrate of ␤ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the K m for the peptide. The molecular mechanisms involved in signal transduction of G protein-coupled receptors are best understood in the visual system where rhodopsin serves as the "receptor" for light (1) and the ␤-adrenergic pathway in which the ␤-adrenergic receptor (␤AR) 1 binds catecholamines (2, 3). A feature common to both model systems as well as many other G protein receptors is the diminished responsiveness with time to a signal of equal intensity. This phenomenon is known as desensitization (4) and exhibits both an agonist-specific and nonspecific pattern. Rapid, agonist-specific desensitization of rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR) occurs in response to the phosphorylation of the receptor by the enzymes rhodopsin kinase and the ␤-adrenergic receptor kinase (␤ARK) (5). Rhodopsin kinase and ␤ARK are members of a family known as G proteincoupled receptor kinases (GRKs). A common feature to the GRK family of kinases is multi-site phosphorylation of receptor substrates in response to agonist occupancy (6). The relationship between agonist occupancy and receptor phosphorylation by GRKs is key to the specificity of the desensitization process, while other kinases such as protein kinase A and C play a role in nonspecific or heterologous desensitization. Two possible mechanisms could explain the enhanced phosphorylation of the activated form of the receptor by kinases of the GRK family. First, receptor occupancy may induce a conformational change exposing potential phosphorylation sites previously sequestered from the kinase. Alternatively, interaction of the kinase with the agonist-bound form of the receptor could result in enhanced catalytic activity of the kinase. The bulk of the experimental evidence supports the latter hypothesis (7-9). In addition to the enhanced catalytic activity of GRKs in the presence of agonist-occupied receptor, GRK2 and GRK3 activity is also increased by heterotrimeric G protein ␤␥ subunits (10 -13). The potential for finely controlled desensitization by the interplay of receptors and ␤␥ subunits is an exciting possibility given the evidence for dual regulation of GRK2 and GRK3 by these proteins (14). While G prot...

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

“…The resulting pellet was resuspended in a small volume of 20 mM Tris-HCl, pH 7.5, 2 mM EDTA. In order to assess the efficiency of reconstitution, saturation binding with [ 125 I]iodopindolol was performed as described previously (13).…”

“…Phosphatidylinositol-3,4-phosphate (PIP 2 ) also binds to GRK2, although reports have varied as to whether PIP 2 serves to activate or inhibit GRK activity (12)(13)(14)(15)(16). Harlan et al (12) have suggested, based on analogy with other PH domains, that GRK2 may bind phospholipids through determinants in the N-terminal half of the PH domain, although this hypothesis has not been tested.…”

“…A group of negatively charged membrane phospholipids, including phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid, and cardiolipin, has also been shown to promote the activity and membrane localization of GRK2 (12)(13)(14)(15)(16). Phosphatidylinositol-3,4-phosphate (PIP 2 ) also binds to GRK2, although reports have varied as to whether PIP 2 serves to activate or inhibit GRK activity (12)(13)(14)(15)(16).…”

Peptide substrates for G protein‐coupled receptor kinase 2

“…The RESA peptide (RRREEEEESAAA) is the only small peptide so far reported to function as a substrate for GRK2 [10], and it has been used to investigate the biochemical characteristics of phosphorylation by GRK2 [11][12][13][14]. Note that phosphorylation sites are indicated by bold italic type; single-letter abbreviations for amino acids are defined in the footnote of Table 1.…”

“…Reported GRK2 phosphorylation targets include b-tubulin [11,15,16], synucleins (a and b) [17], Nedd4 [18], Nedd4-2 [18], b2-adrenergic receptor [19], epithelial Na + channels [20], phosducin [21], rhodopsin [22], ribosomal protein P2 [23], downstream regulatory element antagonist modulator (DREAM) [24] (MAPK) [25], phosphodiesterase c (PDEc) [26], Smad2 [27], ezrin/ radixin [28,29], and platelet-derived growth factor receptor-b (PDGFR-b) [30]. Phosphorylation of substrates by GRK2 can be enhanced in the presence of G protein bc subunits and negatively charged phospholipids (e.g., phosphatidylserine) [13,16,31].…”