mTORC1- and mTORC2-interacting proteins keep their multifunctional partners focused

The mammalian target of rapamycin, best known as mTOR, is a phylogenetically conserved serine/threonine kinase that controls life-defining cellular processes such as growth, metabolism, survival, and migration under the influence of multiple interacting proteins. Historically, the cellular activities blocked by rapamycin in mammalian cells were considered the only events controlled by mTOR. However, this paradigm changed with the discovery of two signaling complexes differentially sensitive to rapamycin, whose catalytic component is mTOR. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to growth factors and nutrients via the phosphorylation of p70S6K and 4EBP1; while the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of AKT, respectively. Although mTORC2 kinase activity is not inhibited by rapamycin, hours of incubation with this antibiotic can impede the assembly of this signaling complex. The direct mechanism by which mTORC2 leads to cell migration depends on its interaction with P-Rex1, a Rac-specific guanine nucleotide exchange factor, while additional indirect pathways involve the intervention of PKC or AKT, multifunctional ubiquitous serine/threonine kinases that activate effectors of cell migration upon being phosphorylated by mTORC2 in response to chemotactic signals. These mTORC2 effectors are altered in metastatic cancer. Numerous clinical trials are testing mTOR inhibitors as potential antineoplasic drugs. Here, we briefly review the actions of mTOR with emphasis on the controlling role of mTORC1 and mTORC2-interacting proteins and highlight the mechanisms linked to cell migration.     

Rac1 regulates the activity of mTORC1 and mTORC2 and controls cellular size

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously.     

Lack of a significant role of P-Rex1, a major regulator of macrophage Rac1 activation and chemotaxis,in atherogenesis

BackgroundRho GTPases are known to play important roles in regulating multiple cellular processes that include cell polarization and migration. Among these Rho GTPases, Rac has been shown to be essential for F actin formation and cell migration. P-Rex1 is a guanine nucleotide exchange factor (GEF) that was previously found to mediate the activation of Rac2, but not Rac1, in mouse neutrophils.ObjectivesHere we examined the role of P-Rex1 in mouse macrophages and atherogenesis.Methods and resultsPBD (p21 binding domain) pull down assay was performed to compare the Rac1 activation in WT and P-Rex1-deficient macrophage. In addition, transwell assay was conducted to compare chemotaxis of WT and P-Rex1-deficient macrophage. We found that P-Rex1 is a major Rac1 regulator in mouse macrophages as its deficiency significantly compromises macrophage chemotaxis, superoxide production (SOD), and Rac1 activation in response to chemoattractants. The potential role of P-Rex1 in atherogenesis is also investigated by transferring P-Rex1-deficient bone marrow cells to LDLR deficient mice. Contrary to our prediction, P-Rex1 deficiency did not alter atherogenesis, suggesting chemoattractant-induced macrophage migration may not have a significant role in atherogenesis.ConclusionsP-Rex1 is one of the major GEFs in macrophage regulating Rac1 activation and chemotaxis.     

Role of guanine nucleotide exchange factor P-Rex-2b in sphingosine 1-phosphate-induced Rac1 activation and cell migration in endothelial cells

Endothelial cell (EC) migration has an important role in angiogenesis. Sphingosine-1 phosphate (S1P) stimulates EC migration via activation of Gi proteins. In this study, we characterized a mouse guanine nucleotide exchange factor (GEF) P-Rex2b for its regulation by Gbetagamma and PI3K and its role in S1P-induced Rac1 activation and cell migration in ECs. We found that co-expression of Gbetagamma or an active form of PI3K (PI3K(AC)) with P-Rex2b increased the SRE.Luciferase (SRE.L) reporter gene activity that can be stimulated by the Rho family of small GTPases including Rac1. Co-expression with P-Rex2b of Gbetagamma and PI3K(AC) or wild type PI3Kgamma that can be activated by Gbetagamma led to further increases in the reporter gene activity. Together with the finding that co-expression of Gbetagamma and/or PI3K(AC) increased the levels of active Rac1, we conclude that P-Rex2b is a Rac GEF that can be regulated by Gbetagamma and PI3K. Additionally, we demonstrated that Gbetagamma interacted with P-Rex2b, probably through P-Rex2b sequences at the PH domain and that the DEP and PDZ domains of P-Rex2b exerted an inhibitory effect on P-Rex2b's activity because their deletion increased the SER.L reporter gene activity. Furthermore, we found that P-Rex2b is involved in S1P-induced Rac1 activation and cell migration in ECs because siRNA-mediated suppression of P-Rex2b expression in ECs-diminished Rac1 activation and cell migration in response to S1P. Therefore, P-Rex2b is a physiologically significant Rac1 GEF that has an important role in the regulation of EC migration.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.     

P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils

Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils, but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions. Here, we characterized P-Rex1, a Gbetagamma and PIP(3)-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants, for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2(S17N) than Rac1(S17N). In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.     

TSC1 Controls Distribution of Actin Fibers through Its Effect on Function of Rho Family of Small GTPases and Regulates Cell Migration and Polarity

The tumor-suppressor genes TSC1 and TSC2 are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of TSC1 and TSC2 form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a Tsc1-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.     

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.     

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs,GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.     

Hold on tightly

Signaling regulated by Rho small GTPases plays a pivotal role in cell migration, cell attachment to substratum or to their neighbors among other functions. Concerted efforts have focused on understanding how different GTPases are activated by specific stimuli and which regulator is responsible for the spatio-temporal control of their activity at particular intracellular sites. We have recently described the role of a scaffold protein, Ajuba, in adherens junction maintenance via direct stabilization of activated small GTPase Rac1 at cell–cell contacts. Ajuba binds to both active and inactive forms of Rac1. Upon junction formation, Rac1 activation initiates a positive feedback loop leading to Ajuba phosphorylation and Ajuba-mediated retention of activated Rac1 at junctions. Thus, cytoskeletal proteins may have a dual role to provide a scaffolding platform and dynamically modulate small GTPases function at a specific place, irrespective of their ability to interact with active and inactive forms. Here we discuss similar mechanisms via which cytoskeletal proteins can facilitate cellular processes downstream of Rho proteins by increasing their affinity to activated GTPases.     

Coronin1 Proteins Dictate Rac1 Intracellular Dynamics and Cytoskeletal Output

Rac1 regulates lamellipodium formation, myosin II-dependent contractility, and focal adhesions during cell migration. While the spatiotemporal assembly of those processes is well characterized, the signaling mechanisms involved remain obscure. We report here that the cytoskeleton-related Coronin1A and -1B proteins control a myosin II inactivation-dependent step that dictates the intracellular dynamics and cytoskeletal output of active Rac1. This step is signaling-branch specific, since it affects the functional competence of active Rac1 only when forming complexes with downstream ArhGEF7 and Pak proteins in actomyosin-rich structures. The pathway is used by default unless Rac1 is actively rerouted away from the structures by upstream activators and signals from other Rho GTPases. These results indicate that Coronin1 proteins are at the center of a regulatory hub that coordinates Rac1 activation, effector exchange, and the F-actin organization state during cell signaling. Targeting this route could be useful to hamper migration of cancer cells harboring oncogenic RAC1 mutations.     

Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance [1-3]. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance [4-7]. Using yeast two-hybrid screening and in vitro interaction assays, we show that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induced similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. Our findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration.     

Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.     

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.     

p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho

The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.     

Serum-activated assembly and membrane translocation of an endogenous Rac1:effector complex

Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras [1], and in a variety of cellular processes [2]. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.     

Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma cells

Members of the Rho family of small GTPases, such as Rho and Rac, are required for actin cytoskeletal reorganization during the migration of carcinoma cells. Phosphodiesterases are necessary for this migration because they alleviate cAMP-dependent protein kinase (PKA)-mediated inhibition of RhoA (O'Connor, K. L., Shaw, L. M., and Mercurio, A. M. (1998) J. Cell Biol. 143, 1749-1760; O'Connor K. L., Nguyen, B.-K., and Mercurio, A. M. (2000), J. Cell Biol. 148, 253-258). In this study, we report that the migration of breast and squamous carcinoma cells toward either lysophosphatidic acid or epidermal growth factor involves not only phosphodiesterase activity but also cooperative signaling from PKA. Furthermore, we demonstrate that Rac1 activation in response to chemoattractant or beta(1) integrin clustering is regulated by PKA and that Rac1 is required for this migration. Also, we find that beta(1) integrin signaling stimulates the rapid and transient activation of PKA. A novel implication of these findings is that carcinoma cell migration is controlled by cAMP-dependent as well as cAMP inhibitory signaling mechanisms.     

Rac activation: P-Rex1 - a convergence point for PIP(3) and Gbetagamma?

P-Rex1, a novel Rac activator, has been identified in the first biochemical purification of a guanine nucleotide exchange factor for GTPases of the Rho family. P-Rex1 is synergistically activated by PIP(3) and Gbetagamma and may act as a coincidence detector for these signaling molecules.     

P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.