G Protein-Coupled Receptor Kinases: From ... - The FASEB Journal

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*Department of Pharmacology, Vanderbilt University Medical Center, Nashville, ... Region, Russia; and §Faculty of Biology, St. Petersburg State University, St.
The FASEB Journal • FASEB SRC Commentary

G Protein-Coupled Receptor Kinases: From Molecules to Diseases Eugenia V. Gurevich,*,1 Richard T. Premont,† and Raul R. Gainetdinov

‡,{,§

*Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; † Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA; and ‡Italian Institute of Technology, Genova, Italy; {Skolkovo Institute of Science and Technology, Skolkovo, Moscow Region, Russia; and §Faculty of Biology, St. Petersburg State University, St. Petersburg, Russia

Attendees of the FASEB Science Research Conference: G Protein-Coupled Receptor Kinases: From Molecules to Diseases (June 8–13, 2014) in Steamboat Springs, CO, USA.

THE 2014 FASEB SCIENCE Research Conference (SRC) on G Protein-Coupled Receptor Kinases: From Molecules to Diseases focused on recent advances in understanding the biochemistry of G protein-coupled receptor kinases (GRKs), their physiologic functions, and roles in pathologic conditions. GRKs are key regulators that, together with arrestins, determine the rate and extent of homologous desensitization of G protein-coupled receptors (GPCRs). GPCRs mediate responses to hormones and neurotransmitters in multiple tissues and cell types and are targeted by many drugs. Therefore, proteins regulating GPCR signaling are critical for a variety of diseases and represent important therapeutic targets. Recent seminal discoveries attracted attention to novel aspects of GRK biology, such as regulation of non-GPCR receptors and control of GPCR signaling in a phosphorylation-independent manner. This meeting brought together researchers studying varying aspects of GRK biology in this rapidly expanding field, from structural biologists to physiologists and physicianscientists. The discussions broadened our understanding of fundamental functions of these exciting proteins and facilitated collaborations. The interdisciplinary nature of the 0892-6638/15/0029-0361 © FASEB

meeting stems from appreciation that only joint effort of investigators studying the GRK action in different model systems will overcome remaining challenges in the GRK field and will allow researchers to fully exploit the potential of GRKs as therapeutic targets. GPCRs are the largest superfamily of signaling proteins, with several hundred subtypes in all mammalian species. These receptors share a 7-transmembrane span structure and transmit the signal of agonist binding to the cell surface receptor in uniform manner via activation of intracellular heterotrimeric G proteins, as the name implies. The GPCR family occupies a position of extraordinary importance in physiology and medicine, not only because GPCRs control every aspect of physiological function but also because GPCRs are targeted by a large percentage of clinically used drugs. It is not surprising that GPCR signaling is under strict control via multiple mechanisms. One 1

Correspondence: Department of Pharmacology, Vanderbilt University, 2200 Pierce Ave., PRB417C, Nashville, TN 37232. E-mail: [email protected] doi: 10.1096/fj.14-263657ufm

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such critical mechanism controlling the intensity and duration of the GPCR signaling is homologous desensitization. As soon as a receptor is activated by a bound ligand to activate its cognate G protein, the opposing process begins, aimed at shutting down the ability of the receptor to respond to continuous stimulation by the ligand. This mechanism serves not only to prevent overstimulation but also to achieve low background and high temporal resolution of the cellular signaling events. The classic model of homologous desensitization posits that agonist-activated receptors are phosphorylated by a GRK, which promotes high-affinity binding of an uncoupling protein termed arrestin. Arrestin shields the cytoplasmic surface of the receptor, precluding further G protein activation. Arrestin bound to the receptor further promotes receptor internalization by virtue of its interaction with clathrin and adapter protein-2 (AP2), the main components of clathrin-coated pits, thereby initiating receptor sorting and recycling. Arrestins also redirect GPCR signaling to G protein-independent pathways via arrestin interaction with numerous protein partners. Thus, GRKs and arrestins play key roles in regulating GPCR responsiveness to agonists via desensitization, resensitization, and arrestinmediated signaling. The GRK-dependent receptor phosphorylation is the first and rate-limiting step in this cascade. Thus, GRKs are the key regulatory proteins determining the rate and extent of desensitization and internalization of GPCRs and, consequently, impacting the intensity and duration of the GPCR signaling. They are also critical regulators of signaling pathways mediated through arrestins, as well as traditional heterotrimeric G proteins. Considering that GPCR dysregulation is a critical contributor to many diseases, GRKs play roles in a multitude of biologic processes and the pathogenesis of many diseases.

This structural arrangement clearly separates GRKs from all other AGC kinases. Functionally, the most striking feature of GRKs is their exquisite selectivity for activated GPCRs. With regard to GRKs, the receptor plays the role of both a substrate and allosteric activator stimulating its own phosphorylation. The molecular mechanism of such activation remains one of the most fascinating mysteries in GRK biology. Although the functional importance of GRKs in regulating GPCR signaling became apparent early on, studies on GRK biology lagged behind that of other kinases. The studies of the GRK biochemistry were confined for a long time to in vitro experimentation using visual receptor rhodopsin or b2-adrenergic receptor as a substrate. Phosphorylation of specific receptors by GRK isoforms remains poorly studied to this day. Considering that 2 GRKs are confined to the retina and 1 confined mainly to the testis, the fact that there are only 4 widely expressed GRKs available to deal with hundreds of GPCRs gave rise to the notion that GRK isoforms have no receptor specificity, interpreted exclusively as inherent biochemical specificity. The notion is still popular even today, in spite of the data obtained in cells and in knockout mouse strains that clearly contradict it. The presence of multiple potential phosphorylation sites in many GPCRs makes it difficult to determine the sites that are actually phosphorylated by each GRK isoform, particularly because GRKs, unlike many other kinases, lack clear preferred recognition sequences. Lack of comprehensive knowledge of the receptor phosphorylation pattern by each GRK isoform remains an impediment to understanding the regulation of the receptor functions by the GRKs. GRKS COME OF AGE

GRKS—THE FORGOTTEN ONES Phosphorylation of rhodopsin, a prototypical GPCR, upon its activation by light was described first as early as 1972, and the first GRK—named “opsin kinase” or “rhodopsin kinase” (systematic name GRK1) that selectively phosphorylates active rhodopsin—was identified soon after (1). The role of rhodopsin phosphorylation in its rapid deactivation was demonstrated first in 1980, and in a few years, a similar mechanism was shown for b2-adrenergic receptor and then for many GPCRs. The first kinase that could phosphorylate specifically activated b-adrenergic receptors—first named the b2-adrenergic receptor kinase (now GRK2)—was identified by Jeffrey Benovic in the laboratory of Robert Lefkowitz in 1986 (2), the same year in which it was realized that both rhodopsin and b2adrenergic receptor are members of the same large GPCR superfamily. A total of 7 GRK isoforms, GRK1 through GRK7, have been identified and classified into 3 distinct subfamilies. GRKs belong to the large family of AGC kinases that includes such familiar kinases such as protein kinases A and C. However, GRKs stand apart within this family structurally as well as functionally. The structure of GRKs is intriguing; the kinase domain is inserted into the regulator of G-protein signaling (RGS) homology domain, splitting it into the two unequal N- and C-terminal parts. 362

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Recently, several important advances have been made in the field of GRK biology. First, the crystal structures of representative members of the 3 subfamilies of GRKs have been solved (GRK1; GRK2 in complex with Gbg, and with both Gaq and Gbg; GRK6; and GRK6 in an active-like conformation), which provide a critical foundation for structure-function studies (3–5). Second, truly novel data on the role of GRKs have become available in GPCR signaling and trafficking, in receptor specificity and interaction of GRK isoforms with GPCRs, and in mechanisms of GRK subcellular targeting, regulation, and degradation (6, 7). Third, we now know that GRKs play an important role in regulating signaling via phosphorylation of several types of non-GPCR receptors (8, 9). Fourth, it is now clear that GRKs also can regulate GPCR and non-GPCR signaling in a phosphorylation-independent manner. One such mechanism involves RGS homology domain, the distinctive structural feature of the GRK family, which can bind and sequester active Gaq, thereby reducing Gaq-mediated signaling (10). A second mechanism involves pleckstrinhomology domain found in GRK2/3 that not only mediates the recruitment of these kinases to the plasma membrane via its interaction with Gbg but is also capable of regulating the Gbg-dependent signaling (11). Novel data on the physiologic significance of these regulatory modes are emerging. Fifth, GRKs have been implicated in multiple disease states, including several neural diseases, heart

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GUREVICH ET AL.

diseases, hypertension, insulin resistance, inflammation, and several forms of cancer. GRK2 has been considered the prime therapeutic target for congestive heart failure for years, but now there are specific GRK2/3 inhibitors available for experiments and, hopefully, for treatment. Most of these developments have occurred within the last 5– 10 years. The new findings have expanded the repertoire of GRK functions from pure desensitization-related role to the general regulatory role in a variety of signaling processes critical for human health (12). GRK-FOCUSED CONFERENCE—THE FIRST EVER There has never been a specialized conference on GRK biology or biochemistry. GRK-related subjects are usually discussed within the framework of meetings focusing on general GPCR pharmacology (e.g., International Catecholamine Symposium; Molecular Pharmacology Gordon Research Conference; and Second Messengers Gordon Conference) or in bits and pieces at the larger meetings sponsored by the Society for Neuroscience, American Heart Association, or Endocrine Society. Considering the abundance of exciting new data in the GRK field, the time has come to have a multidisciplinary meeting covering various aspects of GRK biology. This meeting was meant to bring GRK researchers with different perspectives, from structure to behavior, together for the first time. The FASEB SRC on “G Protein-Coupled Receptor Kinases: From Molecules to Diseases” was held on June 8–13, 2014. GRKologists from around the world met in Steamboat Springs, Colorado, in the first-ever international meeting that focused entirely on these important regulators of the GPCR signaling. Virtually, every laboratory actively working with GRKs was represented among speakers and attendees. The organizers were Eugenia V. Gurevich (Vanderbilt University, Nashville, TN, USA), Raul R. Gainetdinov (Italian Institute of Technology, Genoa, Italy), and Richard T. Premont (Duke University, Durham, NC, USA). The meeting began on Sunday with a keynote address by Marc Caron (Duke University), who provided a historical overview of how GRKs were discovered and initially characterized. Bringing his work into the present day, Dr. Caron described the use of cell type-specific conditional knockouts to examine the regulation of dopamine receptors in the striatal locomotor circuit and dopamine-dependent behaviors by GRK isoforms. Over the next 4.5 days, we held 9 sessions organized by topics. Each session consisted of four 25-min talks and two to three 15-min short talks. Ample time was available for questions and discussion. The main themes included the following: 1) GRK structure and mechanism of activation; 2) GRK function in the cardiovascular pathology; 3) GRK regulation, expression, and degradation; 4) GRK-dependent regulation of receptor signaling; 5) GRK-dependent phosphorylation of nonGPCR substrates; 6) phosphorylation-independent regulation of signaling by GRKs; 7) GRK in neuronal functions; and 8) GRKs in model organisms. The rules of FASEB conferences prohibit the disclosure of original findings presented at the conference. However, some of the studies discussed at the meeting had been published previously or have been published since the conference. Therefore, we can mention some of the FASEB SRC G PROTEIN-COUPLED RECEPTOR KINASES

communications highlighting the key findings in the GRK field, as well as the emerging trends. John Tesmer (University of Michigan, Ann Arbor, MI, USA) led the discussion of structure, activation, and inhibition of GRKs. Multiple GRK structures from the Tesmer laboratory have identified many commonalities with other AGC kinases (a two-lobe structure where ATP and substrate bind in the cleft between the lobes in a closed, active conformation) as well as unique details on how GRKs bind lipids and G protein subunits. Current efforts of the Tesmer laboratory and that of others are focused on identifying the structural elements of GRKs critical for the GRK binding to and activation by receptors and on structural changes in the GRK molecule upon binding to the receptor. Such structural information can guide the development of isoformselective modulators of GRK activity that are currently lacking. As the meeting progressed, however, it became exceedingly obvious that the structural information based exclusively on the GRK interaction with GPCRs would be insufficient. Many speakers presented evidence of the GRK ability to phosphorylate and regulate the activity of non-GPCR receptors. Neil Freedman (Duke University) described studies of GRK5 phosphorylation of the plateletderived growth factor b receptor in vascular smooth muscle cells, whereas Leonard Girnita (Karolinska Institutet, Stockholm, Sweden) described GRK phosphorylation of insulin-like growth factor type 1 receptor. Therefore, the cases of GRK-dependent phosphorylation of receptors other than traditional 7-transmembrane GPCRs that were treated until recently as curious exceptions now appear quite common, which suggests that GRK-dependent regulation is not restricted to GPCRs but extends to a much wider variety of receptors. The speakers discussed an astonishing range of pathological conditions in which deregulation of the GRK function plays the key role. The role of GRK2 in the congestive heart failure was established many years ago, and large body of evidence has been collected on the beneficial effect of GRK2 inhibition in this condition, with several recent studies presented at the meeting. GRK2, a ubiquitous and highly expressed GRK isoform, appears to be involved in heart failure, vascular pathology, hypertension, and the functions of the adrenal medulla. In addition to these rather traditional areas in the GRK research, an abundance of studies on the roles of GRKs in various forms of cancer came as a surprise. It turned out that in cancerous cells GRKs regulate a multitude of processes such as cell proliferation, survival, and motility via phosphorylationdependent and independent mechanisms. Another strong theme that emerged at the meeting was the roles of GRKs in inflammation and inflammatory processes in diseases. These studies supported the model of GRKs as a central node in cellular signaling networks as presented by Federico Mayor Jr. (Universidad Autonoma de Madrid, Madrid, Spain). It became increasingly clear as the meeting progressed that the role of GRKs in regulating cellular signaling is so much broader than envisioned previously. We feel that the role GRKs play in the neural functions deserves a special mention, although, admittedly, our own interests biased our perception. Nevertheless, in the nervous system, signaling processes are, perhaps, more diverse and intense than in any other tissue. The data presented at the conference demonstrated that GRKs in neurons are 363

positioned at the signaling crossroads. GRKs are critical for the normal function of the nervous system and could serve as important therapeutic targets in neural diseases, such as chronic pain, drug addiction, or neurodegenerative diseases. It was obvious that GRKs in the brain are grossly understudied, with even the most obvious and basic questions having no answers. Most speakers emphasized very specific regulation of GPCRs by GRK isoforms in different neuronal populations determined by cellular complement of GRK isoforms. However, the expression of GRKs in specific neuronal types is poorly described, and changes in the GRK expression in the brain regions in pathological conditions and in response to drugs are also essentially unknown. In general, the GRK field would greatly benefit from the availability of inhibitors specific for GRK isoforms. Several presenters described their effort to develop such inhibitors, giving hope that GRKs will truly become “druggable” in the near future. It is clear that much more effort is needed to fully understand the role each GRK isoform in function and dysfunction of the nervous and other systems. GRKs are critically important regulatory proteins with huge therapeutic potential, which needs to be fully explored and exploited. THE TRIAL IS OVER—WHAT IS NEXT? The meeting was attended by scientists from the United States, many European countries, Japan, China, and Israel. With very few exceptions attributed to unforeseen circumstances, every invited speaker attended and stayed the entire time, which reflected high enthusiasm for the meeting within the GRK community. The funding for the meeting was provided by the U.S. National Institutes of Health National Institute on Drug Abuse, FASEB, Eli Lilly, and AbbVie. We are grateful for their support, which allowed us to partially compensate the attendees for expenses of the meeting. The feedback from the attendees overwhelmingly demonstrated that the scientific program of the conference was a success. Presented material served to inform of new findings as well as to stimulate their own ideas. The general enthusiasm for the meeting led to the decision at the business meeting to make the conference a regular affair. Three exceptional scientists—unquestionable leaders in the GRK field—were elected to serve as organizers for the next GRK conference in 2017: Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA, USA), Federico Mayor Jr. (Universidad Autonoma de Madrid), and John Tesmer (University of Michigan). One issue discussed by the attendees in the postmeeting assessment was whether it would be advisable to incorporate studies of arrestins in addition to GRKs. Because in the receptor desensitization process GRKs lay groundwork for arrestins, this is a natural thought. However, the opinions were divided. Some attendees believed that arrestin-related studies could overwhelm GRK-specific subjects. It will be up to the

newly elected organizers to devise the scientific program for the next meeting. What will they decide? The authors thank Kristen Hagy, the FASEB conference manager, for her hard work and help in the preparation of the meeting material and in ensuring a well-organized meeting. They also thank Nan Nootenboom for her enthusiastic help and support on site in ensuring that the meeting proceeded smoothly and that everyone had a great time. The work was supported by the U.S. National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (Grant NS065868) and NIH National Institute on Drug Abuse (Grant DA030103) (to E.V.G.), U.S. Department of Defense Medical Research and Development Program (Grant DM102564) (to R.T.P.), and the Russian Science Foundation (Grant 14-25-00065) (to R.R.G.).

REFERENCES 1. Weller, M., Virmaux, N., and Mandel, P. (1975) Light-stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin. Proc. Natl. Acad. Sci. USA 72, 381–385 2. Benovic, J. L., Mayor, F., Jr., Somers, R. L., Caron, M. G., and Lefkowitz, R. J. (1986) Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase. Nature 321, 869–872 3. Boguth, C. A., Singh, P., Huang, C. C., and Tesmer, J. J. (2010) Molecular basis for activation of G protein-coupled receptor kinases. EMBO J. 29, 3249–3259 4. Lodowski, D. T., Pitcher, J. A., Capel, W. D., Lefkowitz, R. J., and Tesmer, J. J. (2003) Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma. Science 300, 1256–1262 5. Tesmer, V. M., Kawano, T., Shankaranarayanan, A., Kozasa, T., and Tesmer, J. J. (2005) Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex. Science 310, 1686–1690 6. Nogu´es, L., Salcedo, A., Mayor, F., Jr., and Penela, P. (2011) Multiple scaffolding functions of beta-arrestins in the degradation of G protein-coupled receptor kinase 2. J. Biol. Chem. 286, 1165–1173 7. Salcedo, A., Mayor, F., Jr., and Penela, P. (2006) Mdm2 is involved in the ubiquitination and degradation of G-proteincoupled receptor kinase 2. EMBO J. 25, 4752–4762 8. Zheng, H., Worrall, C., Shen, H., Issad, T., Seregard, S., Girnita, A., and Girnita, L. (2012) Selective recruitment of G proteincoupled receptor kinases (GRKs) controls signaling of the insulin-like growth factor 1 receptor. Proc. Natl. Acad. Sci. USA 109, 7055–7060 9. Freedman, N. J., Kim, L. K., Murray, J. P., Exum, S. T., Brian, L., Wu, J. H., and Peppel, K. (2002) Phosphorylation of the plateletderived growth factor receptor-b and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization. J. Biol. Chem. 277, 48261–48269 10. Dhami, G. K., and Ferguson, S. S. (2006) Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis. Pharmacol. Ther. 111, 260–271 11. Raveh, A., Cooper, A., Guy-David, L., and Reuveny, E. (2010) Nonenzymatic rapid control of GIRK channel function by a G protein-coupled receptor kinase. Cell 143, 750–760 12. Gurevich, E. V., Tesmer, J. J., Mushegian, A., and Gurevich, V. V. (2012) G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. Pharmacol. Ther. 133, 40–69

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