G protein-coupled receptor kinases: More than just kinases and not only for GPCRs
Introduction
Signaling via G protein-coupled receptors (GPCR) is terminated by a remarkably uniform two-step mechanism: a GPCR kinase (GRK) phosphorylates the active receptor, converting it into a target for high affinity binding of arrestin. Bound arrestin shields the cytoplasmic surface of the receptor, precluding G protein binding and activation (Wilden, 1995, Krupnick et al., 1997).
Phosphorylation of rhodopsin, a prototypical GPCR, upon its activation by light was first described in 1972 (Bownds et al., 1972, Kühn and Dreyer, 1972). Soon thereafter “opsin kinase” (modern name GRK11), which selectively phosphorylates active rhodopsin, was identified (Weller et al., 1975). The first clear evidence that rhodopsin phosphorylation is necessary for its rapid deactivation was presented in 1980 and led to the hypothesis that this mechanism may also regulate hormone receptors (Liebman & Pugh, 1980). Within a few years, this idea was confirmed for β2-adrenergic receptor (β2AR) (Stadel et al., 1983, Sibley et al., 1985) and later for many others (reviewed in Carman & Benovic, 1998). The demonstration of sequence similarity between the β2AR and rhodopsin in 1986 (Dixon et al., 1986) led to the recognition of the family of G protein-coupled receptors (GPCRs), of which rhodopsin is a founding member. Also in 1986, a kinase that could phosphorylate activated β-adrenergic receptors (βARK; modern name GRK2) was identified (Benovic et al., 1986b). This enzyme could also phosphorylate rhodopsin in a light-dependent manner (Benovic et al., 1986a). Phosphorylation of rhodopsin facilitates the binding of another protein termed arrestin (called 48-kDa protein at the time), which physically blocks further signaling by the receptor to heterotrimeric G proteins (Wilden et al., 1986). Demonstration that desensitization of the β2AR requires a homolog of arrestin (Benovic et al., 1987) firmly established the paradigm of two-step GPCR inactivation, which was later shown to apply to the majority of GPCRs (Carman and Benovic, 1998, Gurevich and Gurevich, 2004, Gurevich and Gurevich, 2006b). The cloning of GRK2 in 1989 suggested that it belongs to a distinct lineage of eukaryotic Ser/Thr protein kinases (Benovic et al., 1989a) that are a subclass of the AGC kinase group (Manning et al., 2002). In rapid succession, the members of this family expanded to include βARK2 (GRK3) (Benovic et al., 1991), GRK4 (Ambrose et al., 1992), GRK5 (Kunapuli & Benovic, 1993), and GRK6 (Benovic & Gomez, 1993). Cone specific GRK7 (Hisatomi et al., 1998, Weiss et al., 1998) completed the set of vertebrate GRKs.
The expression of mammalian GRK1 and GRK7 is largely limited to vertebrate rod and cone photoreceptors although both are also present in pinealocytes (Somers and Klein, 1984, Zhao et al., 1997, Zhao et al., 1999, Pugh and Lamb, 2000). Virtually every mammalian cell expresses several isoforms of non-visual GRKs from early embryonic development. GRK4 is expressed at high levels only in testis (Premont et al., 1996). In addition, GRK4 expression was detected in proximal tubule cells in kidneys, where GRK4α and GRK4γ variants reportedly regulate the signaling of D1 and D3 dopamine receptors (Felder et al., 2002, Villar et al., 2009). GRK4 is also expressed in the brain (Sallese et al., 2000b) and uterus myometrium (Brenninkmeijer et al., 1999). In the rat brain, four GRK isoforms, GRKs 2, 3, 5, and 6, are found as early as embryonic day 14 (Gurevich et al., 2004). Unfortunately, the information about the cell-specific expression of GRK isoforms is limited. We mostly know their distribution at the tissue level. The cellular complement of GRK isoforms may prove to be the most important determinant of specificity in GRK function. For example, both GRK1 and GRK2 efficiently phosphorylate light-activated rhodopsin, but GRK2 does not perform this function in GRK1 knockout mice.
The importance of the GRK-mediated signal shutoff is best illustrated in the visual system, where the lack of GRK1 or sites for GRK phosphorylation on rhodopsin leads to the loss of photoresponses, photoreceptor degeneration, and blindness in mice and night blindness in humans (Chen, Makino, et al., 1995, Yamamoto et al., 1997, Khani et al., 1998, Chen et al., 1999, Zhang et al., 2005, Hayashi et al., 2007, Song et al., 2009, Fan et al., 2010). In other cell types, the results are not as dramatic, except in development. In Drosophila, Gprk2,2 an ortholog of GRK4/5/6, is required for wing morphogenesis (Molnar et al., 2007), egg morphogenesis, and embryogenesis (Schneider & Spradling, 1997). Knockdown of Grk2 in zebrafish embryos induces early developmental arrest (Jiang et al., 2009), and knockout of GRK2 in mice is embryonic lethal due to abnormal formation of the heart (Jaber et al., 1996). This lethality stems from general, albeit undefined, role of GRK2 in embryogenesis, rather than specific role in the heart development, because mice with GRK2 ablation specific to the cardiac myocytes develop normally (Matkovich et al., 2006).
We know about structure and function of GRKs a lot less than these proteins deserve, considering that GRKs critically influence the function of most GPCRs, which are the targets of a large percentage of clinically used drugs (Gruber et al., 2010). Many issues are far from resolved. GRK specificity towards particular receptor subtypes is one important unanswered question. As mammals have only five non-visual GRKs and >800 GPCRs (Gruber et al., 2010), there are hundreds of GPCRs per GRK. It follows that each GRK must have the ability to phosphorylate many different receptors. However, neither the level of receptor specificity nor actual preference for particular GPCRs of non-visual GRKs is clear. We also need to fully describe how active GPCRs activate GRKs, which would be greatly facilitated by a structure of a receptor–GRK complex. This would define the full receptor footprint on the GRK and provide greater insight into the mechanism of kinase activation. GRKs phosphorylate many non-GPCR substrates, but it remains unknown whether proteins other than GPCRs can activate GRKs.
GRKs 2 and 5 have long been considered promising therapeutic targets for cardiac diseases (Penela et al., 2006). However, there has been little research regarding their value as therapeutic targets for other conditions. We believe that GRKs, by virtue of their regulatory nature, hold a great promise for therapy of disorders involving an imbalance in GPCR signaling. However, a better understanding of their structure and function is a prerequisite for successful therapeutic intervention.
Section snippets
Structural organization of G protein-coupled receptor kinases
Based on sequence similarity and gene structure, vertebrate GRKs are classified into three subfamilies: GRK1 comprising GRK1 (rhodopsin kinase) and GRK7 (cone kinase), GRK2 comprising GRK2 and 3, and GRK4 comprising GRK4, 5, and 6 (Premont et al., 1999). All GRKs are multi-domain proteins (Fig. 1) consisting of ~25-residue N-terminal region unique to the GRK family of kinases, followed by the regulator of G protein signaling (RGS) homology domain (RH) (Siderovski et al., 1996), and a Ser/Thr
Subcellular targeting of G protein-coupled kinase isoforms
The various GRK subfamilies employ several distinct mechanisms that bring them to or retain them at the membrane, where their integral membrane substrates GPCRs are found (Fig. 1). Visual subtypes have characteristic CaaX motif on the C-terminus for prenylation: GRK1 is farnesylated (Inglese et al., 1992a), whereas GRK7 is geranylgeranylated (Hisatomi et al., 1998). The association of GRKs 1 and 7 with the membrane is mediated by C-terminal prenylation. Therefore, visual GRKs “search” for
Mechanism of activation of G protein-coupled receptor kinases by active G protein-coupled receptors
The ability to phosphorylate active GPCRs was the first GRK function to be discovered. Receptor phosphorylation by itself can decrease G protein coupling (Wilden, 1995) and enables high-affinity binding of arrestin, which stops G protein-mediated signaling by blocking the cytoplasmic surface of the receptors (Krupnick et al., 1997). The most striking feature distinguishing GRKs from other kinases is that their activity depends on the functional state of the target: GRKs effectively
G protein-coupled receptor kinases phosphorylate non-G protein-coupled receptor substrates
An intriguing development in recent years has been a discovery of the ability of GRKs to interact with a variety of proteins other than GPCRs and in many cases to phosphorylate them (Table 1). The data extend the repertoire of pathways whose signaling is controlled by GRKs via phosphorylation of various signaling components. The list of non-GPCR substrates now includes single transmembrane domain tyrosine kinases (PDGFRβ), single transmembrane domain serine/threonine kinases, death receptors,
Proteins regulated by G protein-coupled receptor kinases in phosphorylation-independent manner
GRKs have been reported to regulate several signaling proteins via direct interaction that does not require kinase activity (Table 2). It is not unusual for enzymes to perform scaffolding functions in addition to or instead of their enzymatic activity. GRKs, particularly GRK2 and 3, are fairly large multidomain proteins, and it is conceivable that they could interact with a multitude of proteins via different domains (Pronin et al., 1997, Lodowski et al., 2003, Tesmer et al., 2005). As the
Regulation of G protein-coupled receptor kinases
The expression level, as well as activity of most enzymes in the cell is tightly regulated. GRKs are no exception. As described above, the best-known mechanism of GRK regulation is via direct binding to active GPCRs. However, this is just one of several established regulatory mechanisms.
G protein-coupled kinase isoforms — more of the same?
No review of GRKs would be complete without discussion of receptor specificity of GRK isoforms. The situation with GRKs resembles that with arrestins (Gurevich & Gurevich, 2006a) in that there are too few of them to be strictly receptor specific. Thus, it is often assumed that GRK isoforms are “nonspecific” towards GPCRs. However, the issue is not as simple as it appears. There are just too many GRK isoforms preserved over millions of years of vertebrate and mammalian evolution to believe that
Physiological and pathological roles of G protein-coupled kinase isoforms
In spite of the obvious importance of GRKs for the regulation of the GPCR signaling, their physiological functions remain poorly understood. Although the vertebrate GRK family is not large – only 7 members – it is functionally quite diverse, with two members, GRKs 1 and 7, playing specialized roles in photoreceptor cells and others ubiquitously expressed throughout the body obviously fulfilling multiple needs. To sort out all these complex interlinked functions played by individual GRK isoforms
Conclusions and future prospects
GRKs are well-established regulators of signaling and trafficking of GPCRs, the most numerous class of cell surface receptors targeted by about half of clinically used drugs (Jacoby et al., 2006). Recent findings show that GRKs also regulate a number of other GPCR-independent signaling pathways intimately involved in many vital cellular functions. Despite their obvious importance, GRKs are under-appreciated as drug targets and therapeutic tools.
In order to take full advantage of GRKs as
Conflict of interest statement
The authors declare that there are no conflicts of interests.
Acknowledgment
This work was supported by NIH grants NS065868 (EVG), HL071818 (JJGT), EY011500, GM077561, GM081756 (VVG), and by Stowers Institute for Medical Research (ARM).
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