Receptor tyrosine kinases are signaling intermediates of G protein-coupled receptors

G protein-coupled receptors (GPCRs) can utilize receptor tyrosine kinases (RTKs) to mediate important cellular responses such as proliferation, differentiation and survival. Recent advances in the field suggest that GPCR-induced transactivation of RTKs might be important for diseases such as cancer and cardiac hypertrophy. Depending on the receptor and cell type, GPCR signaling involves activation of several different RTKs. By activating different subsets of RTKs, GPCRs can fine-tune their effects on target cells. Furthermore, RTK-independent signaling pathways also initiated by GPCRs may modify the biological read out of the transactivated RTKs. This review focuses on the mechanisms how GPCRs and intracellular messengers elicit transactivation of different RTKs and the resulting different biological responses.     

Signal transduction pathways of G protein-coupled receptors and their cross-talk with receptor tyrosine kinases: lessons from bradykinin signaling

G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.     

Cyclin-dependent protein kinases as therapeutic targets in cardiovascular disease

Excessive cell proliferation is thought to contribute to the development of neointimal lesions during the pathogenesis of atherosclerosis, restenosis and vessel bypass graft failure. Since cell cycle progression requires the sequential activation of cyclin-dependent kinases (CDKs), through their association with regulatory subunits dubbed cyclins, therapeutic strategies via CDK inhibition may reduce the burden of vascular proliferative disease. Some of these approaches may rely on the use of pharmacological CDK inhibitors that target the ATP-binding pocket of the catalytic site of CDK. Others are based on the overexpression of members of the family of CDK inhibitory proteins (CKIs), which associate with and inhibit the activity of CDK/cyclin holoenzymes. In this review, animal studies that document the efficacy of pharmacological agents and gene therapy approaches directly targeting CDK/cyclins for the treatment of vascular proliferative disease are discussed. Recent patent applications in this field are also analysed.     

Transgenic mice targeting the heart unveil G protein-coupled receptor kinases as therapeutic targets

GRKs critically regulate betaAR signaling via receptor phosphorylation and the triggering of desensitization. In the heart, betaARs control the chronotropic, lusitropic, and inotropic responses to the catecholamine neurotransmitters, norepinephrine and epinephrine. Signaling through cardiac betaARs is significantly impaired in many cardiovascular disorders, including congestive heart failure. betaARK1 (also known as GRK2) is the most abundant GRK in the heart, and it is increased in several cardiovascular diseases associated with impaired cardiac signaling and function, suggesting that this molecule could have pathophysiological relevance in the setting of heart failure. The ability to manipulate the mouse genome has provided a powerful tool to study the physiological implications of altering GRK activity and expression in the heart. Recent studies in several different mouse models have demonstrated that betaARK1 plays a key role not only in the regulation of myocardial signaling, but also in cardiac function and development. Moreover, studies have shown that targeting the activity of GRKs, especially betaARK1, appears to be a novel therapeutic strategy for the treatment of the failing heart. Gene therapy technology makes it possible, beyond what is possible in the mouse, to directly test in larger animals whether betaARK1 inhibition in the setting of disease will improve the function of the compromised heart, and this methodology has also lead to compelling results. These genetic approaches or the development of small molecule inhibitors of betaARK1 and GRK activity may advance therapeutic options for heart disease.     

Adiponectin and cardiovascular disease: mechanisms and new therapeutic approaches

Adiponectin is an abundant plasma protein secreted from adipocytes. Its role in energy homeostasis is well-known, including the regulation of hydrocarbons and lipids metabolism as well as the improvement of insulin resistance. It has been thought to be a key molecule in the development of type 2 diabetes mellitus and metabolic syndrome, which are epidemiological targets for preventing cardiovascular disease. In addition to beneficial metabolic effects, adiponectin seems to have anti-inflammatory, anti-atherosclerotic and vasoprotective actions. Furthermore, adiponectin affects signalling in myocardial cells and exerts beneficial actions on the heart after pressure overload and ischemia-reperfusion injury. The ability of adiponectin to reduce insulin resistance in conjunction with its antiinflammatory and cardioprotective properties makes this adipocytokine a promising therapeutic target. On clinical interest, agents that enhance endogenous adiponectin production or action have potential for the treatment of cardiovascular disease. Management strategies that increase adiponectin levels include weight reduction, Mediterranean diet, thiazolidinediones, antihypertensive and lipid lowering drugs. Current knowledge on the main actions of adiponectin and therapeutic approaches for cardiovascular disease is summarized in this review.     

Heterodimerization of g protein-coupled receptors: specificity and functional significance

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.     

G protein-coupled receptor kinases regulate metabotropic glutamate receptor 5 function and expression

Metabotropic glutamate receptors (mGluR) serve important neuromodulatory roles at glutamatergic synapses to shape excitatory neurotransmission. Recent evidence indicates that the desensitization of mGluRs is an important determinant in regulating the functions of these receptors. The present results demonstrate that G protein-coupled receptor kinases (GRKs), which are known to regulate the desensitization of many G protein-coupled receptors, regulate both the expression and function of mGluR5 in a heterologous expression system. This regulatory event is limited to members of the GRK2 family since GRK4 family members do not elicit the same effects on mGluR5. Kinase activity is shown to be required for GRK-mediated regulation of mGluR5. Furthermore, the ability of GRK2 to regulate mGluR5 is dependent, at least in part, on the presence of threonine 840 in the carboxyl terminus of mGluR5. These studies identify novel roles for GRKs in regulating mGluR5 that may serve to further shape the function of these receptors in neurotransmission.     

Ubiquitination of g protein-coupled receptors: functional implications and drug discovery

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of signaling receptors and control a vast array of physiological responses. Modulating the signaling responses of GPCRs therapeutically is important for the treatment of various diseases, and discovering new aspects of GPCR signal regulation is critical for future drug development. Post-translational modifications are integral to the regulation of GPCR function. In addition to phosphorylation, many GPCRs are reversibly modified with ubiquitin. Ubiquitin is covalently attached to lysine residues within the cytoplasmic domains of GPCRs by ubiquitin ligases and removed by ubiquitin-specific proteases. In many cases, ubiquitin functions as a sorting signal that facilitates trafficking of mammalian GPCRs from endosomes to lysosomes for degradation, but not all GPCRs use this pathway. Moreover, there are distinct types of ubiquitin conjugations that are known to serve diverse functions in controlling a wide range of cellular processes, suggesting broad roles for GPCR ubiquitination. In this review, we highlight recent studies that illustrate various roles for ubiquitin in regulation of GPCR function. Ubiquitination is known to target many GPCRs for lysosomal degradation, and current studies now indicate that basal ubiquitination, deubiquitination, and transubiquitination of certain GPCRs are important for controlling cell surface expression and cellular responsiveness. In addition, novel functions for ubiquitin in regulation of GPCR dimers and in mediating differential GPCR regulation induced by biased agonists have been reported. We will discuss the implications of these new discoveries for ubiquitin regulation of GPCR function in the context of drug development.     

Single transmembrane spanning heterotrimeric g protein-coupled receptors and their signaling cascades

Heptahelical of serpentine receptors such as the adrenergic receptors are well known to mediate their actions via heterotrimeric GTP-binding proteins. Likewise, receptors that traverse the cell membrane once have been shown to mediate their biological actions by activating several different mechanisms including stimulation of their intrinsic tyrosine kinase activities or the kinase activities of other proteins. Some of these single transmembrane receptors have an intrinsic guanylyl cyclase activity and can stimulate the cyclic GMP second messenger system; however, over the last few years, several studies have shown the involvement of heterotrimeric GTP-binding proteins in mediating signals that eventually culminate in the biological actions of single transmembrane spanning receptors and proteins. These receptors include the receptor tyrosine kinases that mediate the actions of growth factors such as epidermal growth factor, insulin, insulin-like growth factor as well as receptors for atrial natiuretic hormone or the zona pellucida protein (ZP3) and integrins. In this review, the significance of the coupling of the single transmembrane spanning receptors to G proteins has been highlighted by providing several examples of the concept that signaling via these receptors may involve the activation of multiple signaling cascades.     

Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors

Importance of the field: Axl and/or Mer expression correlates with poor prognosis in several cancers. Until recently, the role of these receptor tyrosine kinases (RTKs) in development and progression of cancer remained unexplained. Studies demonstrating that Axl and Mer contribute to cell survival, migration, invasion, metastasis and chemosensitivity justify further investigation of Axl and Mer as novel therapeutic targets in cancer.

Areas covered in this review: Axl and Mer signaling pathways in cancer cells are summarized and evidence validating these RTKs as therapeutic targets in glioblastoma multiforme, NSCLC, and breast cancer is examined. A discussion of Axl and/or Mer inhibitors in development is provided.

What the reader will gain: Potential toxicities associated with Axl or Mer inhibition are addressed. We propose that the probable action of Mer and Axl inhibitors on cells within the tumor microenvironment will provide a therapeutic opportunity to target both tumor cells and the stromal components that facilitate disease progression.

Take home message: Axl and Mer mediate multiple oncogenic phenotypes and activation of these RTKs constitutes a mechanism of chemoresistance in a variety of solid tumors. Targeted inhibition of these RTKs may be effective as anti-tumor and/or anti-metastatic therapy, particularly if combined with standard cytotoxic therapies.     

Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases

Because of their central role in the cellular response to growth factors, assays of MAP kinase activity are commonly used in pharmaceutical screening efforts aimed at detecting chemical modifiers of growth regulatory pathways. As our understanding of the complexity of signal transduction networks expands, however, it is becoming apparent that previously unappreciated temporal and contextual factors have profound effects on MAP kinase function. This is exemplified by recent studies of the regulation of the ERK1/2 MAP kinase cascade by GPCRs. Depending on receptor and cell type, GPCR stimulation of ERK1/2 can reflect a heterogenous array of signaling events. Activation of second messenger-dependent protein kinases and cross talk between GPCRs and receptor or nonreceptor tyrosine kinases can all induce ERK1/2 activation. Furthermore, a growing body of data indicates that the mechanism of ERK1/2 activation is a major determinant of ERK1/2 function. Activation of a nuclear pool of ERK1/2 as a consequence of cross talk between GPCRs and growth factor receptor tyrosine kinases may provide a mitogenic stimulus. In contrast, activation of ERK1/2 in localized pools on the membrane or confined to endosomal vesicles through the utilization of focal adhesions or beta-arrestins as "scaffolds" may spatially constrain ERK1/2 activity and favor the phosphorylation of nonnuclear ERK substrates. Findings such as these suggest that screening strategies that use single readouts of MAP kinase activity or function are likely to miss important signaling events, and point to the need for a multidimensional approach to MAP kinase-based screening efforts.     

Eph receptor tyrosine kinases in tumor and tumor microenvironment

Eph receptors are a unique family of receptor tyrosine kinases (RTK) that play critical roles in embryonic patterning, neuronal targeting, and vascular development during embryogenesis. In adults, Eph RTKs and their ligands, the ephrins, are frequently overexpressed in a variety of cancers and tumor cell lines, including breast, prostate, non-small cell lung and colon cancers, melanomas, and neuroblastomas. Unlike traditional oncogenes that often function only in tumor cells, recent data show that Eph receptors mediate cell-cell interaction both in tumor cells and in tumor microenvironment, namely the tumor stroma and tumor vasculature. As such, Eph RTKs represent attractive potential targets for drug design, as targeting these molecules could attack several aspects of tumor progression simultaneously. This review will focus on recent advances in dissecting the role of Eph RTKs in tumor cells, tumor angiogenesis, and possible contribution to trafficking of inflammatory cells in cancer.     

Cannabinoid CB1 receptors transactivate multiple receptor tyrosine kinases and regulate serine/threonine kinases to activate ERK in neuronal cells

BACKGROUND AND PURPOSE

Signalling networks that regulate the progression of cannabinoid CB₁ receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB₁ receptor-stimulated ERK phosphorylation in a neuronal cell model.

EXPERIMENTAL APPROACH

Murine N18TG2 neuronal cells were used to analyse the effect of specific protein kinase and phosphatase inhibitors on CB₁ receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation.

KEY RESULTS

The time-course of CB₁ receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0–5 min) maximal ERK phosphorylation is mediated by CB₁ receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires G_i/oβγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5–10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB₁ receptor-stimulated, ligand-independent, transactivation of multiple RTKs.

CONCLUSIONS AND IMPLICATIONS

The complex expression of CB₁ receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signalling. RTK and PKA pathways may provide routes to novel CB₁-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Light-activated receptor tyrosine kinases: Designs and applications

Receptor tyrosine kinases (RTKs) are a large and essential membrane receptor family. The molecular mechanisms and physiological consequences of RTK activation depend on, for example, ligand identity, subcellular localization, and developmental or disease stage. In the past few years, genetically-encoded light-activated RTKs (Opto-RTKs) have been developed to dissect these complexities by providing reversible and spatio-temporal control over cell signaling. These methods have very recently matured to include highly-sensitive multi-color actuators. The new ability to regulate RTK activity with high precision has been recently harnessed to gain mechanistic insights in subcellular, tissue, and animal models. Because of their sophisticated engineering, Opto-RTKs may only mirror some aspects of natural activation mechanisms but nevertheless offer unique opportunities to study RTK signaling and physiology.