Structural insights into G protein-coupled receptor kinase function

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The atomic structure of a protein can greatly advance our understanding of molecular recognition and catalysis, properties of fundamental importance in signal transduction. However, a single structure is incapable of fully describing how a protein functions, particularly when allostery is involved. Recent advances in the structure and function of G protein-coupled receptor (GPCR) kinases (GRKs) have concentrated on the mechanism of their inhibition by small and large molecules. These studies have generated a wealth of new information on the conformational flexibility of these enzymes, which opens new avenues for the development of selective chemical probes and provides deeper insights into the molecular basis for activation of these enzymes by GPCRs and phospholipids

Introduction

G protein-coupled receptor (GPCR) kinases (GRKs) initiate the homologous desensitization of activated GPCRs through the phosphorylation of specific sites within the cytoplasmic loops and carboxy-terminal tails of the receptors [1]. These covalent modifications help to recruit arrestins, which uncouple the GPCRs from heterotrimeric G proteins and targets them for internalization. There are 7 mammalian GRKs grouped into 3 subfamilies (GRK1, GRK2, and GRK4) [2] (Figure 1). Atomic structures representing each subfamily (GRK1 [3], GRK2 [4, 5], and GRK6 [6, 7••]) in various ligand-bound states are now available. These structures establish that the conserved structural core of GRKs is comprised of a protein kinase domain inserted into a loop of a regulator of G protein signaling homology (RH) domain [8]. The RH domain serves as an intramolecular scaffold that maintains the small lobe of the kinase domain in a state that is competent to phosphorylate activated GPCRs. Consequently, the kinase domain, although closely related to those of protein kinases A, G and C (AGC kinases), does not require phosphorylation on its activation loop for full activity. GRKs, however, retain the C-terminal extension of the kinase domain characteristic of the AGC kinase family, which contributes residues to the active site cleft. Although this element is not fully ordered in most GRK structures, mutations in this region in GRK2 [9] and GRK1 [10] are known to dramatically inhibit the phosphorylation of receptor and soluble substrates, consistent with the idea that this element serves to regulate kinase activity as it does in other AGC kinases [11]. The first ∼20 amino acids of GRKs are highly conserved and critical for GPCR and phospholipid-stimulated autophosphorylation. However, this region is disordered in most GRK structures reported to date, clouding interpretation of its molecular role.

This review highlights recent advances in our molecular understanding of GRK function. The most recent structural studies have emphasized the conformational variability of the GRK kinase domain, an understanding of which will likely be key for the development of selective chemical probes. Some of the observed conformational changes observed have also provided much needed structural insight into how these enzymes are recognized and activated by agonist occupied GPCRs and/or phospholipids.

Section snippets

Inhibiting the GRKs

Various GRKs are known to play roles in human disease [12]. GRK2 and GRK5 stand out due to their well characterized roles in heart failure and cardiac hypertrophy [13, 14, 15, 16, 17]. One of the most selective inhibitors of GRK2 known is βARKct (Figure 1), a fragment corresponding to the 222 C-terminal residues of GRK2 [13, 18], which can be administered via adeno-associated virus gene delivery and improves contractile performance in both small and large animal models of heart failure [14, 19

The N-terminal conundrum

The importance of the extreme N-terminal region of GRKs for function was first recognized when antibodies directed against the N-terminus of GRK1 blocked the phosphorylation of rhodopsin but not peptide substrates [29]. Deletion of the first 14 amino acids or mutations in this span of GRK5 leads to loss of phospholipid-dependent autophosphorylation, and the corresponding 14-mer peptide from GRK5 could inhibit GRK5 but not GRK2-dependent phosphorylation of rhodopsin. Phosphorylation of tubulin,

Structure and location of the anionic phospholipid binding site

Each GRK subfamily is localized to the membrane via different mechanisms (Figure 1): GRK1 subfamily members are prenylated, GRK2 subfamily members bind to Gβγ, which is itself prenylated, and GRK4 subfamily members contain two basic regions: immediately following the N-terminal helix and at their C-terminus, which in some isoforms is also palmitoylated [34]. In addition to membrane localization, anionic phospholipids are required for full activity of GRK2 and GRK4 subfamily members [34]. The PH

Conclusions

Recent structural analyses of GRKs have greatly expanded our molecular understanding of this family of enzymes. These atomic models serve as templates for structure-based rational drug design and have spawned new hypotheses regarding the molecular nature of their interactions with activated receptors and phospholipids. The identification of paroxetine as an effective inhibitor of GRK2 in vitro, in cells, and presumably in live animals strongly suggests that more potent GRK2-selective molecules

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This work was supported by the National Institute of Health grants HL071818 and HL086865 (to JJGT) and the American Heart Association grant N014938 (to KTH).

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