RGS proteins, GRKs, and beta-arrestins modulate G protein-mediated signaling pathways in asthma.

Asthma is a highly prevalent disorder characterized by chronic lung inflammation and reversible airways obstruction. Pathophysiological features of asthma include episodic and reversible airway narrowing due to increased bronchial smooth muscle shortening in response to external and host-derived mediators, excessive mucus secretion into the airway lumen, and airway remodeling. The aberrant airway smooth muscle (ASM) phenotype observed in asthma manifests as increased sensitivity to contractile mediators (EC50) and an increase in the magnitude of contraction (Emax); collectively these attributes have been termed "airways hyper-responsiveness" (AHR). This defining feature of asthma can be promoted by environmental factors including airborne allergens, viruses, and air pollution and other irritants. AHR reduces airway caliber and obstructs airflow, evoking clinical symptoms such as cough, wheezing and shortness of breath. G-protein-coupled receptors (GPCRs) have a central function in asthma through their impact on ASM and airway inflammation. Many but not all treatments for asthma target GPCRs mediating ASM contraction or relaxation. Here we discuss the roles of specific GPCRs, G proteins, and their associated signaling pathways, in asthma, with an emphasis on endogenous mechanisms of GPCR regulation of ASM tone and lung inflammation including regulators of G-protein signaling (RGS) proteins, G-protein coupled receptor kinases (GRKs), and ß-arrestin.

Grk1b and Grk7a Both Contribute to the Recovery of the Isolated Cone Photoresponse in Larval Zebrafish.

Purpose: To define the functional roles of Grk1 and Grk7 in zebrafish cones in vivo. Methods: Genome editing was used to generate grk7a and grk1b knockout zebrafish. Electroretinogram (ERG) analyses of the isolated cone mass receptor potential and the b-wave were performed in dark-adapted zebrafish using a paired flash paradigm to determine recovery of cone photoreceptors and the inner retina after an initial flash. In addition, psychophysical visual response was measured using the optokinetic response (OKR). Results: ERG analysis demonstrated that deletion of either Grk1b or Grk7a in zebrafish larvae resulted in modestly lower rates of recovery of the isolated cone mass receptor potential from an initial flash compared to wildtype larvae. On the other hand, grk1b-/- and grk7a-/- larvae exhibited a b-wave recovery that was similar to wildtype larvae. We evaluated the OKR and found that deletion of either Grk1b or Grk7a leads to a small decrease in temporal contrast sensitivity and alterations in visual acuity. Conclusions: For the first time, we demonstrate that Grk1b and Grk7a both contribute to visual function in larval zebrafish cones. Since the difference between wildtype and each knockout fish is modest, it appears that either GRK is sufficient for adequate cone visual function.

Differential regulation of ß2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle.

Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) [e.g. ß2-adrenoceptor (ß2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that ß2AR and A2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for ß2AR- and A2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged ß2AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A2BR was regulated by GRK5, but not GRK2. ß2AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A2BR-AC signalling and attenuated A2BR desensitization, while ß2AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.

The Role of G Protein-coupled Receptor Kinases in Cancer.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. Emerging evidence demonstrates that signaling through GPCRs affects numerous aspects of cancer biology such as vascular remolding, invasion, and migration. Therefore, development of GPCR-targeted drugs could provide a new therapeutic strategy to treating a variety of cancers. G protein-coupled receptor kinases (GRKs) modulate GPCR signaling by interacting with the ligand-activated GPCR and phosphorylating its intracellular domain. This phosphorylation initiates receptor desensitization and internalization, which inhibits downstream signaling pathways related to cancer progression. GRKs can also regulate non-GPCR substrates, resulting in the modulation of a different set of pathophysiological pathways. In this review, we will discuss the role of GRKs in modulating cell signaling and cancer progression, as well as the therapeutic potential of targeting GRKs.

G protein-coupled receptor kinases (GRKs) in tumorigenesis and cancer progression: GPCR regulators and signaling hubs.

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.

Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments.

Dendritic Cells Interpret Haptotactic Chemokine Gradients in a Manner Governed by Signal-to-Noise Ratio and Dependent on GRK6.

Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable "roads" ensure optimal guidance in vivo.

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling.

G protein-coupled receptor kinases (GRKs) are classically known for their role in regulating the activity of the largest known class of membrane receptors, which influence diverse biological processes in every cell type in the human body. As researchers have tried to uncover how this family of kinases, containing only 7 members, achieves selective and coordinated control of receptors, they have uncovered a growing number of noncanonical activities for these kinases. These activities include phosphorylation of nonreceptor targets and kinase-independent molecular interactions. In particular, GRK2, GRK3, and GRK5 are the predominant members expressed in the heart. Their canonical and noncanonical actions within cardiac and other tissues have significant implications for cardiovascular function in healthy animals and for the development and progression of disease. This review summarizes what is currently known regarding the activity of these kinases, and particularly the role of GRK2 and GRK5 in the molecular alterations that occur during heart failure. This review further highlights areas of GRK regulation that remain poorly understood and how they may represent novel targets for therapeutic development.

SCF/c-kit transactivates CXCR4-serine 339 phosphorylation through G protein-coupled receptor kinase 6 and regulates cardiac stem cell migration.

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

Aberrant GRK6 promoter methylation is associated with poor prognosis in hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck cancers with high invasiveness and poor prognosis. To identify targeted therapeutics against metastasis, a better understanding of the regulation of HSCC cell invasion is needed. In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein coupled receptors (GPCRs). However, there is little information regarding GRKs expression in HSCC. In the present study, we examined GRK6 expression in HSCC and also assessed the possible cause of its aberrant expression, as well as its clinical significance. Real-time quantitative PCR (qPCR) and western blotting were performed to analyze the expression of GRK6 in HSCC tissues and corresponding non-malignant tissues. Subsequently, paired HSCC and corresponding non-malignant tissues were evaluated for the methylation status of GRK6 gene promoter using methylation-specific PCR (MSP). Furthermore, we investigated the methylation status and the clinicopathological significance of GRK6 in 45 paired HSCC and corresponding non-malignant tissues. The suppression of GRK6 in hypopharyngeal cell line FaDu by GRK6-shRNA lentivirus transfection was utilized to detect the role of GRK6 in hypopharyngeal cancer progression. Our results showed that the expression of GRK6 mRNA and protein was significantly lower in HSCC than in corresponding adjacent non-tumor tissues, and this downregulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 77.8% (35/45) of the HSCC tissues, while it was found in only 42.2% (19/45) of the corresponding non-malignant tissues. GRK6 methylation was related to depth of tumor invasion and TNM stage. Upon treatment with 5-aza-2'-deoxycytidine, GRK6 expression was upregulated in FaDu cells, and cell invasion was signinficantly inhibited. Furthermore, the suppression of GRK6 by shRNA transfection enhanced FaDu cells invasion. Our results indicate that the aberrant methylation of GRK6 gene promoter may underlie its downregulation in HSCC, and may play an important role in the metastasis of HSCC.

[Research on Molecular Mechanisms of Engulfment of Apoptotic Cells].

Apoptotic cells generated during development and immune responses in animals are rapidly engulfed by phagocytes, such as macrophages and dendritic cells. When the engulfment process malfunctions, the apoptotic cells undergo secondary necrosis, which results in the release of noxious cellular components into the extracellular space. Thus, the efficient clearance of apoptotic cells is indispensable for the maintenance of tissue homeostasis; however, the molecular mechanisms underlying the engulfment of apoptotic cells remain largely unknown. To identify the molecules that are involved in this process, we developed a functional screening strategy using a retrovirus cDNA library. Using this assay, we isolated cDNA clones encoding RhoG and Rab5 which enhanced the engulfment of apoptotic cells. In addition, we found that Rac1, which is very similar to RhoG, and Rab5 are necessary for engulfment; their activities were successfully visualized by a combination of fluorescence resonance energy transfer technology with time-lapse imaging techniques. We further determined that G protein-coupled receptor kinase 6 (GRK6), originally identified as a kinase responsible for the desensitization and downregulation of G-protein-coupled receptors, activates Rac1 independent of the two known intracellular engulfment pathways in phagocytes. GRK6-deficient macrophages exhibited impaired phagocytosis of apoptotic cells. Consequently, GRK6-deficient mice developed autoimmune phenotypes such as an increase in the amount of anti-dsDNA in serum and the deposition of immune complexes in the kidney. Thus, our findings contributed to the understanding of the molecular mechanisms that regulate apoptotic engulfment in phagocytes.

ß-adrenergic receptor-mediated cardiac contractility is inhibited via vasopressin type 1A-receptor-dependent signaling.

BACKGROUND: Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased ß-adrenergic receptor (ßAR) responsiveness. This led us to hypothesize that V1AR signaling regulates ßAR responsiveness and in doing so contributes to development of heart failure. METHODS AND RESULTS: Transaortic constriction resulted in decreased cardiac function and ßAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased ßAR ligand affinity, as well as ßAR-induced Ca(2+) mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of ßAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/G protein receptor kinase-dependent manner. CONCLUSIONS: This newly discovered relationship between cardiac V1AR and ßAR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

G protein-coupled receptors in rheumatology.

G protein-coupled receptors (GPCRs) are transmembrane receptor proteins that allow the transfer of signals across the cell membrane. In addition to their physiological role, GPCRs are involved in many pathophysiological processes including pathways relevant in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis. Two-thirds of all currently available drugs target GPCRs directly or indirectly. However, the detailed mechanism of GPCR signalling is still unclear. Selective modification of GPCR-dependent signalling cascades to inhibit disease progression in rheumatic diseases is now being investigated. One approach is to use antibodies against ligands activating GPCRs. However, several GPCRs are known to be activated by only one ligand. In this case, targeting the receptor itself is a promising approach. So far, more information is available on GPCR action in RA as compared with OA, and even less information is available for other rheumatic diseases. Additional research on the role of GPCRs involved in the pathophysiology of rheumatic diseases is required to develop specific therapeutic approaches.

Decreased expression and role of GRK6 in spinal cord of rats after chronic constriction injury.

Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.

Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells.

AIM: To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells. METHODS: MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 µg/mL penicillin/streptomycin in 5% CO2 at 37 °C. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 µg/mL LPS in this experiment. The viability/proliferation of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A2 (PLA2), phospholipase Cß (PLCß), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA2 (p-PLA2), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLCß and GRK was measured using Western blotting analysis. RESULTS: The activity of MS1 cells incubated with different concentrations of LPS for 6 h decreased significantly in the 1 µg/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 µg/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 µg/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell activity when its concentration reached 50 µmol/L compared with the group that received LPS treatment alone, which was consistent with the results obtained from fluorescence staining. The mRNAs levels of AC (4.02 ± 0.14 vs 1.00 ± 0.13), GRK (2.63 ± 0.03 vs 1.00 ± 0.12), p38 MAPK (3.87 ± 0.07 vs 1.00 ± 0.17), PLA2 (3.31 ± 0.12 vs 1.00 ± 0.12), PLCß (2.09 ± 0.08 vs 1.00 ± 0.06) and PTK (1.85 ± 0.07 vs 1.00 ± 0.11) were up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated mRNAs including AC (2.35 ± 0.13 vs 3.87 ± 0.08), GRK (1.17 ± 0.14 vs 2.65 ± 0.12), p38 MAPK (1.48 ± 0.18 vs 4.30 ± 0.07), PLCß (1.69 ± 0.10 vs 2.41 ± 0.13) and PLA2 (1.87 ± 0.11 vs 2.96 ± 0.08) were significantly suppressed by BN52021 except for that of PTK. The level of p-AC (1.11 ± 0.12 vs 0.65 ± 0.08), GRK (0.83 ± 0.07 vs 0.50 ± 0.03), PLCß (0.83 ± 0.16 vs 0.50 ± 0.10) and p-p38 MAPK (0.74 ± 0.10 vs 0.38 ± 0.05) was up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated proteins, including p-AC (0.65 ± 0.15 vs 1.06 ± 0.14), GRK (0.47 ± 0.10 vs 0.80 ± 0.06), PLCß (0.47 ± 0.04 vs 0.80 ± 0.19) and p-p38 MAPK (0.30 ± 0.10 vs 0.97 ± 0.05), was significantly suppressed by BN52021, but p-PLA2 and p-PTK protein level were not suppressed. CONCLUSION: BN52021 could effectively inhibit LPS-induced inflammation by down-regulating the mRNA and protein levels of AC, GRK, p38 MAPK, PLA2 and PLCß in the PAFR signaling pathway.

Circadian regulation of olfaction and an evolutionarily conserved, nontranscriptional marker in Caenorhabditis elegans.

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.

G protein-coupled receptor kinases in cardiovascular disease: why "where" matters.

Cardiac function is mainly controlled by ß-adrenergic receptors (ß-ARs), members of the G protein-coupled receptor (GPCR) family. GPCR signaling and expression are tightly controlled by G protein-coupled receptor kinases (GRKs), which induce GPCR internalization and signal termination through phosphorylation. Reduced ß-AR density and activity associated with elevated cardiac GRK expression and activity have been described in various cardiovascular diseases. Moreover, alterations in extracardiac GRKs have been observed in blood vessels, adrenal glands, kidneys, and fat cells. The broad tissue distribution of GPCRs and GRKs suggests that a keen appreciation of integrative physiology may drive future therapeutic development. In this review, we provide a brief summary of GRK isoforms, subcellular localization, and interacting partners that impinge directly or indirectly on the cardiovascular system. We also discuss GRK/GPCR interactions and their implications in cardiovascular pathophysiology.

The complexities of G-protein-coupled receptor kinase function in Hedgehog signaling.

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.