Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2.

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

The Antiresorptive Effect of GIP, But Not GLP-2, Is Preserved in Patients With Hypoparathyroidism-A Randomized Crossover Study.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are gut hormones secreted postprandially. In healthy humans, both hormones decrease bone resorption accompanied by a rapid reduction in parathyroid hormone (PTH). The aim of this study was to investigate whether the changes in bone turnover after meal intake and after GIP- and GLP-2 injections, respectively, are mediated via a reduction in PTH secretion. This was tested in female patients with hypoparathyroidism given a standardized liquid mixed-meal test (n = 7) followed by a peptide injection test (n = 4) using a randomized crossover design. We observed that the meal- and GIP- but not the GLP-2-induced changes in bone turnover markers were preserved in the patients with hypoparathyroidism. To understand the underlying mechanisms, we examined the expression of the GIP receptor (GIPR) and the GLP-2 receptor (GLP-2R) in human osteoblasts and osteoclasts as well as in parathyroid tissue. The GIPR was expressed in both human osteoclasts and osteoblasts, whereas the GLP-2R was absent or only weakly expressed in osteoclasts. Furthermore, both GIPR and GLP-2R were expressed in parathyroid tissue. Our findings suggest that the GIP-induced effect on bone turnover may be mediated directly via GIPR expressed in osteoblasts and osteoclasts and that this may occur independent of PTH. In contrast, the effect of GLP-2 on bone turnover seems to depend on changes in PTH and may be mediated through GLP-2R in the parathyroid gland. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Emerging Role of Adhesion GPCRs in Cancer.

Aberrant expression, function, and mutation of G protein-coupled receptors (GPCRs) and their signaling partners, G proteins, have been well documented in many forms of cancer. These cell surface receptors and their endogenous ligands are implicated in all aspects of cancer including proliferation, angiogenesis, invasion, and metastasis. Adhesion GPCRs (aGPCRs) form the second largest family of GPCRs, most of which are orphan receptors with unknown physiological functions. This is mainly due to our limited insight into their structure, natural ligands, signaling pathways, and tissue expression profiles. Nevertheless, recent studies show that aGPCRs play important roles in cell adhesion to the extracellular matrix and cell-cell communication, processes that are dysregulated in cancer. Emerging evidence suggests that aGPCRs are implicated in migration, proliferation, and survival of tumor cells. We here review the role of aGPCRs in the five most common types of cancer (lung, breast, colorectal, prostate, and gastric) and emphasize the importance of further translational studies in this field.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Spatial regulation of GPR64/ADGRG2 signaling by ß-arrestins and GPCR kinases.

Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (ΔNTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ΔNTF and P622 mutants interact with ß-arrestin1 and ß-arrestins2 and are constitutively internalized in steady states. However, only ΔNTF shows exaggerated basal activation of the Gαs -cAMP-CRE signaling cascade. Neither ΔNTF nor P622 shows constitutive activation of the Gα13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ΔNTF and P622 to early endosomes, where ΔNTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with ß-arrestins and the consequent endocytosis and sustained signaling from endosomes.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

Parathyroid-Targeted Overexpression of Regulator of G-Protein Signaling 5 (RGS5) Causes Hyperparathyroidism in Transgenic Mice.

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G-protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT) and that RGS5 can inhibit signaling from the calcium-sensing receptor (CASR). In vivo, we found that RGS5-null mice have abnormally low PTH levels. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain overexpressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence that RGS5 can modulate signaling from CASR and support a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling. © 2019 American Society for Bone and Mineral Research.

RGS4 Overexpression in Lung Attenuates Airway Hyperresponsiveness in Mice.

A cardinal feature of asthma is airway hyperresponsiveness (AHR) to spasmogens, many of which activate G protein-coupled receptors (GPCRs) on airway smooth muscle (ASM) cells. Asthma subtypes associated with allergy are characterized by eosinophilic inflammation in the lung due to the type 2 immune response to allergens and proinflammatory mediators that promote AHR. The degree to which intrinsic abnormalities of ASM contribute to this phenotype remains unknown. The regulators of G protein signaling (RGS) proteins are a large group of intracellular proteins that inhibit GPCR signaling pathways. RGS2- and RGS5-deficient mice develop AHR spontaneously. Although RGS4 is upregulated in ASM from patients with severe asthma, the effects of increased RGS4 expression on AHR in vivo are unknown. Here, we examined the impact of forced RGS4 overexpression in lung on AHR using transgenic (Tg) mice. Tg RGS4 was expressed in bronchial epithelium and ASM in vivo, and protein expression in lung was increased at least 4-fold in Tg mice compared with wild-type (WT) mice. Lung slices from Tg mice contracted less in response to the m3 muscarinic receptor agonist methacholine compared with the WT, although airway resistance in live, unchallenged mice of both strains was similar. Tg mice were partially protected against AHR induced by fungal allergen challenge due to weakened contraction signaling in ASM and reduced type 2 cytokine (IL-5 and IL-13) levels in Tg mice compared with the WT. These results provide support for the hypothesis that increasing RGS4 expression and/or function could be a viable therapeutic strategy for asthma.

Polyclonal origin of parathyroid tumors is common and is associated with multiple gland disease in primary hyperparathyroidism.

BACKGROUND: Parathyroid tumors are mostly considered monoclonal neoplasms, the rationale for focused parathyroidectomy in primary hyperparathyroidism. We reported that flow sorting parathyroid tumor cells and methylation-sensitive polymerase chain reaction (me-PCR) of polymorphic human androgen receptor gene and phosphoglycerate kinase gene alleles in deoxyribonucleic acid reveals that ≤35% of parathyroid tumors are polyclonal. We sought to confirm these findings and assess for clinical relevance. METHODS: Parathyroid tumors from 286 female primary hyperparathyroidism patients were analyzed for clonal status. Tumor clonal status was compared with clinical variables and operative findings. Statistical analysis was performed and significance was established at P < .05. RESULTS: In the study, 176 (62%) patients were informative for human androgen receptor gene and/or phosphoglycerate kinase gene. Assignment of clonal status was made in 119 (68%) tumors, of which 64 (54%) were monoclonal and 55 (46%) were polyclonal. Comparison of tumor clonal status to clinical variables in patients with complete operative data (N = 82) showed that while clinical features were the same between tumor types, patients with polyclonal tumors more often had multiple gland disease (risk ratio 4.066, confidence interval, 1.016-16.26; P = .039) potentially missed at unilateral neck exploration. CONCLUSION: This work confirms that primary hyperparathyroidism is often the result of polyclonal tumors and that parathyroid tumor clonal status may be associated with multiple gland disease.

Reporter gene assays for investigating GPCR signaling.

Luciferase-based assays are applied to evaluate various cellular processes due to their sensitivity and feasibility. The field of GPCR research has also benefited from this enzymatic reaction both in deorphanization campaigns and in delineation of the signaling pathways. Here, we describe the details of this assay in GPCR studies in 96-well format and will provide examples where the assay can show constitutive activity of an orphan GPCR and demonstrate the impact of cell type on the efficacy and potency of ligands.

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium-Sensing Receptor-Mediated Signaling.

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and ß-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca2+ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.

A fungal protease allergen provokes airway hyper-responsiveness in asthma.

Asthma, a common disorder that affects >250 million people worldwide, is defined by exaggerated bronchoconstriction to inflammatory mediators including acetylcholine (ACh), bradykinin and histamine-also termed airway hyper-responsiveness. Nearly 10% of people with asthma have severe, treatment-resistant disease, which is frequently associated with immunoglobulin-E sensitization to ubiquitous fungi, typically Aspergillus fumigatus (Af). Here we show that a major Af allergen, Asp f13, which is a serine protease, alkaline protease 1 (Alp 1), promotes airway hyper-responsiveness by infiltrating the bronchial submucosa and disrupting airway smooth muscle (ASM) cell-extracellular matrix (ECM) interactions. Alp 1-mediated ECM degradation evokes pathophysiological RhoA-dependent Ca(2+) sensitivity and bronchoconstriction. These findings support a pathogenic mechanism in asthma and other lung diseases associated with epithelial barrier impairment, whereby ASM cells respond directly to inhaled environmental allergens to generate airway hyper-responsiveness.

Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation.

BACKGROUND: Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyperresponsiveness (AHR), suggesting that underlying abnormalities in structural cells, such as airway smooth muscle (ASM), contribute to the asthmatic diathesis. Dysregulation of procontractile G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility. OBJECTIVE: We explored the role of a regulator of procontractile GPCR signaling, regulator of G protein signaling 5 (RGS5), in unprovoked and allergen-induced AHR. METHODS: We evaluated GPCR-evoked Ca(2+) signaling, precision-cut lung slice (PCLS) contraction, and lung inflammation in naive and Aspergillus fumigatus-challenged wild-type and Rgs5(-/-) mice. We analyzed lung resistance and dynamic compliance in live anesthetized mice using invasive plethysmography. RESULTS: Loss of RGS5 promoted constitutive AHR because of enhanced GPCR-induced Ca(2+) mobilization in ASM. PCLSs from naive Rgs5(-/-) mice contracted maximally at baseline independently of allergen challenge. RGS5 deficiency had little effect on the parameters of allergic inflammation, including cell counts in bronchoalveolar lavage fluid, mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, induced IL-13 and IL-33 levels were much lower in challenged lungs from Rgs5(-/-) mice relative to those seen in wild-type mice. CONCLUSION: Loss of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. Because it is selectively expressed in ASM within the lung and does not promote inflammation, RGS5 might be a therapeutic target for asthma.

Enhanced effector function of CD8(+) T cells from healthy controls and HIV-infected patients occurs through thrombin activation of protease-activated receptor 1.

Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these 2 processes. The proinflammatory function of thrombin is mediated by activation of protease-activated receptor 1 (PAR-1). We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T-cell motility and production of proinflammatory cytokines.

The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

Regulator of G-protein signaling-5 inhibits bronchial smooth muscle contraction in severe asthma.

Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein-coupled receptor (GPCR)-induced Ca(2+) mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca(2+) mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling-5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca(2+) to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.

Minireview: recent developments in the physiology and pathology of the lysophosphatidylinositol-sensitive receptor GPR55.

Emerging data suggest that off-target cannabinoid effects may be mediated via novel seven-transmembrane spanning/G protein-coupled receptors. Due to its cannabinoid sensitivity, the G protein-coupled receptor 55 (GPR55) was recently proposed as a candidate; however, GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, and the conflicting pharmacology, signaling, and functional data have prevented its classification as a novel cannabinoid receptor. Indeed, the most consistent and potent agonist to date is the noncannabinoid lysophospholipid, lysophosphatidylinositol. Here we present new human GPR55 mRNA expression data, providing supportive evidence of GPR55 expression in a vast array of tissues and cell types. Moreover, we summarize major recent developments in GPR55 research and aim to update the reader in the rapidly expanding fields of GPR55 pharmacology, physiology, and pathology.

Pharmacology, signaling and physiological relevance of the G protein-coupled receptor 55.

According to The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), ∼70 million European adults have consumed cannabis on at least one occasion. Cannabis consumption leads to a variety of psychoactive effects due to the presence of the constituent Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Δ(9)-THC interacts with the endocannabinoid system (ECS), which consists of the seven transmembrane spanning (7TM)/G protein-coupled receptors (GPCRs) CB(1) and CB(2), their respective ligands (endocannabinoids), and enzymes involved in their biosynthesis and degradation. This system plays a critical role in many physiological processes such as learning and memory, appetite control, pain sensation, motor coordination, lipogenesis, modulation of immune response, and the regulation of bone mass. Therefore, a huge effort has been spent trying to fully elucidate the composition and function of the ECS. The G protein-coupled receptor 55 (GPR55) was recently proposed as a novel component of this system; however, its classification as a cannabinoid receptor has been significantly hampered by its complex pharmacology, signaling, and cellular function. GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, but in some experimental paradigms, it is activated by endocannabinoids, phytocannabinoids, and synthetic cannabinoid ligands. However, the most potent compound appears to be a lysophospholipid known as lysophosphatidylinositol (LPI). Here, we provide a comprehensive evaluation of the current pharmacology and signaling of GPR55 and review the proposed role of this receptor in a number of physiological and pathophysiological processes.

Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis.

Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.