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IMMUNOLOGY Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kb in myeloid cells Daniel M. Houslay, 1 * Karen E. Anderson, 1 * Tamara Chessa, 1 * Suhasini Kulkarni, 1 Ralph Fritsch, 2 Julian Downward, 3 Jonathan M. Backer, 4 Len R. Stephens, 1†‡ Phillip T. Hawkins 1†‡ Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110a, p110b, p110g, or p110d) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110b, including direct interaction with Gbg subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110b that selectively blocked the interaction with Gbg and investigated its contribution to the PI3K isoform dependen- cy of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotidebinding protein)coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110b in supporting synergistic PIP 3 formation in response to the coactivation of macrophages by mac- rophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110a, p110b, and p110d downstream of M-CSF alone and a nonredundant role for p110g downstream of C5a alone. This role for p110b completely depended on direct interaction with Gbg, suggesting that p110b transduces GPCR signals in the context of coincident activation by an RTK. The p110b-Gbg interaction was also required for neutrophils to generate reactive oxygen species in re- sponse to the Fcg receptordependent recognition of immune complexes and for their b 2 integrinmediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses. INTRODUCTION Class I phosphoinositide 3-kinases (PI3Ks) are a family of signaling enzymes that synthesize the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and are involved in regulating an array of critical cellular functions, such as growth, proliferation, and cell survival (13). PIP 3 exerts its effects through the recruitment and activation of various effector proteins by specific PIP 3 -binding domains, the best studied being the pleckstrin homology domain, which is present in PDK1 (phosphoinositide- dependent protein kinase 1), Akt1 and Akt2, P-Rex1 (PIP 3 -dependent rac exchanger 1), Grp1 (general receptor for phosphoinositides 1), and BTK (Brutons tyrosine kinase) (4, 5). The class I family of PI3K enzymes con- sists of three class Ia members, which are heterodimers consisting of a p110 catalytic subunit (p110a, p110b, or p110d) combined with one of a shared group of p85regulatory subunits (p85a, its splice variants p50- and p55a, p85b, or p55g), whereas the single class Ib member, PI3Kg, con- sists of a p110g catalytic subunit coupled to either a p101 or p84 regulatory protein (3, 6). The p110a and p110b subunits are ubiquitously present, whereas p110d and p110g are most abundant in hematopoietic cells (7, 8). Class Ia PI3Ks are classically engaged downstream of the activation of receptor tyrosine kinases (RTKs) through the direct binding of two Src homology 2 (SH2) domains in the p85 regulatory subunits with sequence- specific phosphorylated tyrosine residues (phosphotyrosines) in the re- ceptors or their adaptor proteins. PI3Kg activation occurs downstream of G protein (heterotrimeric guanine nucleotidebinding protein)coupled receptor (GPCR) signaling through the binding of Gbg subunits to both p110g and either of the p101 or p84 regulatory subunits (1, 3, 6). Class I PI3Ks can be further regulated through binding to the small guanosine triphosphatases of the Ras, Rho, and Rab families (1, 3, 9). Whereas all class I PI3K family members produce PIP 3 , and class Ia members share high sequence homology and similar domain structures, as well as a common pool of p55 and p85 regulatory subunits, it is becoming increasingly clear that individual PI3K isoforms can play distinct roles in both physiology and pathology (1, 3, 1012). To some extent, the selective involvement of p110g and p110d in cellular responses can be explained by their possession of a unique regulatory subunit or differential tissue dis- tribution, respectively, but the ubiquitous p110a and p110b subunits can also play remarkably selective roles in cell signaling. For example, p110a positively regulates angiogenesis, growth, and anabolic metabolism, and it is a prevalent oncoprotein (1315), whereas p110b is required for efficient platelet activation (16, 17), osteoclast-mediated bone resorption (18), and spermatogenesis (19, 20), and it drives tumorigenesis in cells lacking the phosphatase PTEN (phosphatase and tensin homolog) (21, 22). Detailed investigations of the molecular properties of p110a and p110b have revealed a plausible mechanism by which mutation in the gene encoding p110a more commonly creates hyperactive proteins, but how p110a is selec- tively recruited by several RTKs, particularly receptors for insulin-like 1 Inositide Laboratory, Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB223AT, UK. 2 Department of Hematology and Oncology, Freiburg University Medical Centre, Albert-Ludwigs-Universität, Freiburg, Hugstetter Str. 55 79106, Germany. 3 Signal Transduction Laboratory, Francis Crick Institute, Lincolns Inn Fields, London WC2A 3LY, UK. 4 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Forchheimer 230, Bronx, NY 10461, USA. *These authors contributed equally to this work. These authors contributed equally to this work. Corresponding author. Email: [email protected] (P.T.H.); [email protected] (L.R.S.) RESEARCHARTICLE www.SCIENCESIGNALING.org 16 August 2016 Vol 9 Issue 441 ra82 1 on December 1, 2020 http://stke.sciencemag.org/ Downloaded from

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  • R E S E A R C H A R T I C L E

    I M M U N O L O G Y

    Coincident signals from GPCRs and receptortyrosine kinases are uniquely transduced by PI3Kbin myeloid cellsDaniel M. Houslay,1* Karen E. Anderson,1* Tamara Chessa,1*Suhasini Kulkarni,1 Ralph Fritsch,2 Julian Downward,3

    Jonathan M. Backer,4 Len R. Stephens,1†‡ Phillip T. Hawkins1†‡

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    Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipidmessenger phosphatidylinositol3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth,survival, and movement. This network is overactivated in cancer and inflammation, and there is interest indetermining the PI3K catalytic subunit (p110a, p110b, p110g, or p110d) that should be targeted in differenttherapeutic contexts. Previous studies have defined unique regulatory inputs for p110b, including directinteraction with Gbg subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110b thatselectively blocked the interactionwithGbg and investigated its contribution to the PI3K isoform dependen-cy of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) responses in primarymacrophages and neutrophils.We discovered a unique rolefor p110b in supporting synergistic PIP3 formation in response to the coactivation of macrophages bymac-rophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partiallyredundant roles for p110a, p110b, and p110d downstream of M-CSF alone and a nonredundant role forp110g downstream of C5a alone. This role for p110b completely depended on direct interaction withGbg, suggesting that p110b transduces GPCR signals in the context of coincident activation by an RTK.The p110b-Gbg interaction was also required for neutrophils to generate reactive oxygen species in re-sponse to the Fcg receptor–dependent recognition of immune complexes and for their b2 integrin–mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in thesetwo responses.

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    INTRODUCTION

    Class I phosphoinositide 3-kinases (PI3Ks) are a family of signalingenzymes that synthesize the second messenger phosphatidylinositol3,4,5-trisphosphate (PIP3) and are involved in regulating an array of criticalcellular functions, such as growth, proliferation, and cell survival (1–3).PIP3 exerts its effects through the recruitment and activation of variouseffector proteins by specific PIP3-binding domains, the best studied beingthe pleckstrin homology domain,which is present in PDK1 (phosphoinositide-dependent protein kinase 1), Akt1 and Akt2, P-Rex1 (PIP3-dependent racexchanger 1), Grp1 (general receptor for phosphoinositides 1), and BTK(Bruton’s tyrosine kinase) (4, 5). The class I family of PI3K enzymes con-sists of three class Ia members, which are heterodimers consisting of ap110 catalytic subunit (p110a, p110b, or p110d) combined with one of ashared group of “p85” regulatory subunits (p85a, its splice variants p50-and p55a, p85b, or p55g), whereas the single class Ib member, PI3Kg, con-sists of a p110g catalytic subunit coupled to either a p101 or p84 regulatoryprotein (3, 6). The p110a and p110b subunits are ubiquitously present,whereas p110d and p110g are most abundant in hematopoietic cells (7, 8).

    1InositideLaboratory,BabrahamInstitute,BabrahamResearchCampus,Babraham,Cambridge CB223AT, UK. 2Department of Hematology and Oncology, FreiburgUniversity Medical Centre, Albert-Ludwigs-Universität, Freiburg, Hugstetter Str. 5579106, Germany. 3Signal Transduction Laboratory, Francis Crick Institute, Lincoln’sInn Fields, LondonWC2A3LY,UK. 4Department ofMolecular Pharmacology, AlbertEinstein College of Medicine, Forchheimer 230, Bronx, NY 10461, USA.*These authors contributed equally to this work.†These authors contributed equally to this work.‡Corresponding author. Email: [email protected] (P.T.H.);[email protected] (L.R.S.)

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    Class Ia PI3Ks are classically engaged downstream of the activation ofreceptor tyrosine kinases (RTKs) through the direct binding of two Srchomology 2 (SH2) domains in the p85 regulatory subunits with sequence-specific phosphorylated tyrosine residues (phosphotyrosines) in the re-ceptors or their adaptor proteins. PI3Kg activation occurs downstreamofG protein (heterotrimeric guanine nucleotide–binding protein)–coupledreceptor (GPCR) signaling through the binding of Gbg subunits to bothp110g and either of the p101 or p84 regulatory subunits (1, 3, 6). Class IPI3Ks can be further regulated through binding to the small guanosinetriphosphatases of the Ras, Rho, and Rab families (1, 3, 9).

    Whereas all class I PI3K family members produce PIP3, and class Iamembers share high sequence homology and similar domain structures,aswell as a commonpool of p55 and p85 regulatory subunits, it is becomingincreasingly clear that individual PI3K isoforms can play distinct roles inboth physiology and pathology (1, 3, 10–12). To some extent, the selectiveinvolvement of p110g and p110d in cellular responses can be explained bytheir possession of a unique regulatory subunit or differential tissue dis-tribution, respectively, but the ubiquitous p110a and p110b subunits canalso play remarkably selective roles in cell signaling. For example, p110apositively regulates angiogenesis, growth, and anabolic metabolism, and itis a prevalent oncoprotein (13–15), whereas p110b is required for efficientplatelet activation (16, 17), osteoclast-mediated bone resorption (18), andspermatogenesis (19, 20), and it drives tumorigenesis in cells lacking thephosphatase PTEN (phosphatase and tensin homolog) (21, 22).

    Detailed investigations of the molecular properties of p110a and p110bhave revealed a plausiblemechanismbywhichmutation in the gene encodingp110amore commonly creates hyperactive proteins, but how p110a is selec-tively recruited by several RTKs, particularly receptors for insulin-like

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    growth factor 1 (IGF1), platelet-derivedgrowth factor (PDGF), and epidermalgrowth factor (EGF), is still unclear (23). In contrast, there has been sub-stantial progress in understanding how p110bmight be selectively regulated.Gbg subunits bind to and activate p110b, and this interaction supports sub-stantial, synergistic activation of p110b-p85 dimers by Gbg and phospho-tyrosine peptides in vitro (24, 25). This interaction has been suggested todrive the p110b-dependent activation ofAkt in response toGPCRs invariousmodel cell systems, including mouse embryonic fibroblasts (MEFs) andprimary macrophages (26–28). The Gbg-binding site in p110b is localizedto the C2-helical domain linker, and point mutations (KK532/533DD) and acell-permeable peptide inhibitor have been defined that selectively disruptthis interaction (24). This work has confirmed that direct binding betweenGbg and p110b is essential for the activation of p110b by GPCRs in modelcells and PTEN−/− tumor cell lines (24).

    Furthermore, the Ras-binding domain (RBD) of p110b preferentiallybinds to guanosine triphosphate (GTP)–bound (active) Rac and Cdc42 pro-teins, whereas the RBD of p110a selectively binds to GTP-bound Ras pro-teins (9). Knock-inmutations in theRBDof p110b that disrupt its binding toRac and Cdc42 (S205D/K224A) abolish the p110b-dependent activation ofAkt in response toGPCRstimulation inMEFs and ablate the contribution ofp110b to a mouse model of lung fibrosis (9). In MEFs, GPCRs stimulateguanine nucleotide exchange onRac through the Gbg-dependent regulationof ELMO1-DOCK180 (engulfment and cell motility 1/dedicator of cyto-kinesis 180), thus providing an alternative input by Gbg into the regulationof p110b (9). The helical domain of p110b selectively binds to Rab5, andthis interaction appears to be important for supporting both kinase-dependentand kinase-independent roles for p110b in the control of endocytosis and theinduction of autophagy (29, 30).

    However, the extent to which the individual properties of p110b de-scribed earlier define unique roles for PI3Kb heterodimers in vivo is stillunclear, particularly with respect to its role as a direct transducer of GPCRsignaling. We have attempted to better understand the importance of Gbgbinding to p110b by generating a knock-in mouse strain in which this inter-action is selectively ablated (p110bKK526/527DD). We characterized the acti-vation of PI3K isoforms byGPCRs and RTKs in primarymacrophages andneutrophils isolated from these mice and compared them to cells isolatedfrommicewith a defective p110bRBD (p110bS205D/K224A) or lacking activep110d (p110dD910A) or p110g (p110gKO). We assessed PI3K activity bymeasuring PIP3 directly, thus avoiding the potentially nonlinear readoutsfrom measuring effector responses further downstream in the signalingpathway (for example, the phosphorylation of Akt). We found that stim-ulation of GPCRs alone resulted in the predominant use of p110g, but co-incident stimulation of a GPCR and an RTK resulted in the direct binding ofGbg to p110b to drive a synergistic PIP3 response. These conclusions weresupported bymeasuring additional PI3K-dependent functions of neutrophils,including the production of reactive oxygen species (ROS) in response toimmune complexes, a response that depends on the coincident activationof FcgRs and BLT1 (31).

    RESULTS

    Mice expressing a mutant p110b isoform that cannot bindto Gbg were generatedTo investigate the importance of the direct binding of Gbg to p110b in vivo,we generated knock-in mice (termed b-Gbgmice) with two point mutations(KK526/527DD) in the gene encoding p110b that block this interaction(fig. S1) (24). These mutations affect neither the activation of p85-p110bheterodimers by phosphotyrosine peptides in vitro nor the binding ofRac, Cdc42, or Rab5 to p110b (24, 32). The b-Gbg mice were generated

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    with a self-deleting tAceCre-PGK-EM7-Neo resistance cassette (for thedetailed targeting strategy, see fig. S1B and the Supplementary Materials),and correct targetingwas confirmed by Southern blotting analysis (fig. S2A).The presence of the correct mutations was confirmed by polymerase chainreaction analysis and the sequencing of genomic DNA from mouse tissue(fig. S2, B and C). The b-Gbg mice were healthy and fertile and displayedno overt phenotype. The abundance of p110b in bone marrow–derivedmacrophages (BMDMs) or bone marrow neutrophils (BMNs) from thesemice was unaffected by the introduction of these point mutations (fig. S3).The b-Gbg mice were used in experiments together with b-RBD (RBD-deficient p110b; p110bS205D/K224A) (9), b-KO (p110b-deficient) (31), g-KO(p110g-deficient) (33), and d-KI (kinase-inactive p110d; p110dD910A) (34)mice, as well as with p110b-selective (TGX221) (17), p110d-selective(IC87114) (35), and p110a-selective (BYL719) (36) inhibitors to evaluatethe contributions of individual PI3K isoforms to the generation of PIP3 inresponse to receptor stimulation. We did not perform experiments witha-KO (p110a-deficient) mice because they exhibit a block in early embry-onic development (13).

    The C5a-stimulated generation of PIP3 in macrophages isdriven by p110g, whereas the M-CSF–stimulated generationofPIP3 isdrivenbyacombinationofp110a, p110b, andp110dPrevious work showed that primary BMDMs exhibit substantial class IPI3K–dependent signaling in response to stimulation by macrophagecolony-stimulating factor (M-CSF), which acts through the RTKCSF1R(c-fms), or by C5a, which acts through theGPCRC5AR1 (also known asCD88) (28, 33, 37).We initially established the kinetics and scale of PIP3generation in response to these agonists (the “PIP3 response”) in BMDMsisolated from wild-type mice. Stimulation of serum-starved primaryBMDMswith a submaximal dose of C5a (30 nM; fig. S4A) produced rapidand substantial accumulation of PIP3, which was maximal between 15 and30 s after stimulation (Fig. 1, A and B). Stimulation with an analogousconcentration of M-CSF (30 ng/ml; fig. S4B) generated slightly greateramounts of PIP3 but with very similar kinetics (Fig. 1, C and D). ThePIP3 responses to C5a andM-CSF in BMDMswere thereforewell matchedand offered an excellent opportunity to assess the relative roles of class IPI3K isoforms in mediating GPCR- and RTK-dependent responses, singlyand in combination, within the same cell. The amount of PIP3 generated inresponse to C5a (after 30-s stimulation) in BMDMs lacking p110g (g-KO)was similar to that found in unstimulated wild-type cells; however, neitherthe loss of p110b (b-KO) nor the expression of a kinase-inactive p110d(d-KI) reduced the PIP3 responses of those cells (Fig. 1B). These resultssuggest that p110g plays a dominant, nonredundant role in the generationof PIP3 in response to C5a. In contrast, no single class I PI3K isoformplayed a similarly dominant role in the generation of PIP3 in response toM-CSF (Fig. 1D), at least at 30 s.

    To interrogate further the potential roles of class Ia PI3Ks in the PIP3response to M-CSF, we investigated the effect of inhibiting different com-binations of isoforms. First, p110b and p110dwere inhibited in experimentswith a range of concentrations of TGX221 and both wild-type and d-KIBMDMs. TGX221 has predicted IC50 (median inhibitory concentration)values for p110b and p110d of 7 and 100 nM, respectively (17, 38). Wealso investigated the effect of a single high concentration of the p110d-selective inhibitor IC87114, which has a predicted IC50 for p110d of500 nM (35, 38). Either pharmacological or genetic inhibition of p110dor a combination of the two inhibited theM-CSF–dependent generation ofPIP3 by ~40% compared to that in wild-type cells treated with dimethylsulfoxide (DMSO) (Fig. 1E). Inhibition of p110b by low concentrations ofTGX221 (up to 40 nM) also reduced the amount of PIP3 generated by ~40%,whereas combined inhibition of p110b and p110d (by treating d-KIBMDMs

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    Fig. 1. Characterization of RTK- andGPCR-driven PIP3 responses in BMDMs:6

    centrations of BYL719 for 5 min before being stimulated for 30 s with M-CSF

    Roles of class I PI3Ks. (A to D) Serum-starved BMDMs (1.2 × 10 cells) fromwild-type (WT)micewerestimulatedwith30nMC5a(A)orM-CSF (30ng/ml) (C)for the indicated times, whereas BMDMs (1.2 × 106) from WT (closed bars),b-KO (open bars), d-KI (dark gray bars), or g-KO (light gray bars) mice wereserum-starved overnight before being stimulated for 30 s with either 30 nMC5a (B) orM-CSF (30 ng/ml) (D) or vehicle control (B andD).Where indicated(hatched bars, D), the p110a-selective inhibitor BYL719 (1 mM final) wasadded to WT BMDMs 5 min before the cells were stimulated. (E) BMDMs(1.2 × 106 cells) fromWTor d-KImicewere serum-starved and incubatedwiththe indicated concentrations of TGX221 (WT, black diamonds; d-KI, blacksquares) or 1 mM IC87114 (WT, gray diamonds; d-KI, gray squares) for 5 minbefore being stimulated for 30 s with M-CSF (30 ng/ml). As a control, WTBMDMs were also left unstimulated (gray triangle). (F) BMDMs (1.2 × 106

    cells) fromWT(blackdiamonds)ord-KI (blacksquares)micewereserum-starvedand incubated with vehicle (WT) or 1 mM IC87114 (d-KI) and the indicated con-

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    (30ng/ml). Asacontrol,WTBMDMswerealso left unstimulated (gray triangle).(A to F) Reactions were quenched, lipids were extracted, and the amounts ofPIP3 and PI were quantitated by mass spectrometry (MS) as described inMaterials and Methods. Data are means ± SEM of at least three experiments,each performed in duplicate [except for the WT control in (E), where n = 1],and are expressed as the ratio of the abundance of PIP3 to that of PI (PIP3/PI)to account for any variations in cell input. The following statistical analyses wereperformed. (B) For the comparisons shown: NS, not significant; *P< 0.05, **P <0.01,byone-wayanalysis of variance (ANOVA)withHolm-Sidakpost hoc test.(E) ForM-CSF+0nMTGX221versusM-CSF+ IC87114,P=0.0156 (WT),P=0.7417(d-KI); forM-CSF+0nMTGX221versusM-CSF+TGX221,P

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    with 40 nM TGX221) reduced the amount of PIP3 generated in responseto M-CSF by about 65% (Fig. 1E).

    Next, the effects of increasing concentrations of the p110a-specific in-hibitor BYL719, which has a predicted IC50 in cells of ~100 nM (39), wereinvestigated in both wild-type and d-KI BMDMs pretreated with 40 nMTGX221 (Fig. 1F). Relatively low concentrations of BYL719 resulted ina ~25% further reduction in the amount of PIP3 generated in response toM-CSF in d-KI BMDMs pretreated with TGX221, resulting in an overallinhibition of ~90% (Fig. 1F). Together, these data suggest that all three classIa PI3K isoforms (p110a, p110b, and p110d) are engaged downstream ofthe M-CSF receptor and play substantial roles in supporting PIP3 produc-tion. Our best estimate of the individual relative contributions suggests thateach isoform plays a largely additive role (40% d, 30% b, and 30% a), butthese estimates are subject to significant error. The larger effect of pharma-cological versus genetic inhibition of p110b is assumed to result frompartialcompensation for the loss of p110b protein by other class Ia isoforms; thatis, heterodimers containing either p110a or p110d can occupymore activatingphosphotyrosines in the absence of p110b.

    Coincident activation of macrophages by C5a and M-CSFresults in a synergistic PIP3 response that requires Gbgto bind to p110bSimultaneous stimulation ofwild-type BMDMswith a combination of bothC5a and M-CSF resulted in a synergistic PIP3 response (Fig. 2A). Theamount of PIP3 produced in response to this combined stimulation was~85% greater than that predicted by the sum of the individual responses(costimulation/additive ratio; Fig. 2A, right). This synergistic PIP3 responsewas changed to an additive response by the loss of p110b but not by the lossof p110g or the expression of kinase-inactive p110d (Fig. 2B). The lack ofinvolvement of p110g in the synergistic PIP3 response to the combination ofC5a andM-CSF was particularly striking because there was no measurablePIP3 generated in response toC5a alone in g-KOBMDMs (Fig. 2B, left). Thesynergistic PIP3 response to coincident stimulation by C5a and M-CSF wasalso reduced to an additive response in b-Gbg BMDMs, which essentiallyphenocopied b-KO cells (Fig. 2C). However, substantial synergy was stillseen in b-RBD macrophages (Fig. 2C). These results suggest that the directbinding of Gbg to p110b is required for the p110b-dependent production ofPIP3 in response to stimulation by both C5a and M-CSF.

    Both the C5a- and fMLP-stimulated generation of PIP3 inneutrophils require p110gWeandothers previously showed that p110g plays amajor role in neutrophilactivationbyvariousGi-coupledGPCRs (33,38,40–42).Here,we reevaluatedthe relative involvement of p110b and p110g in GPCR-dependent PIP3generation elicited by either 100 nM C5a or 10 mM fMLP (N-formyl-Met-Leu-Phe). Both C5a and fMLP stimulated very rapid and substantialgeneration of PIP3 by wild-type BMNs, with an initial maximal accumu-lation of PIP3 at 10 s, whereas fMLP also induced a second peak at 60 s(Fig. 3, A and B). The amount of PIP3 generated by wild-type BMNs inresponse toC5awas greater than that in response to fMLP (about threefolddifferent) and was greater than the amount of PIP3 generated by wild-typeBMDMs in response to C5a (about sixfold), but the kinetics of PIP3 pro-duction were similar. The amount of PIP3 that accumulated in g-KO BMNsin response to either fMLPor C5awas reduced by≥90% compared to that inwild-type BMNs (Fig. 3, C and D). In contrast, the amounts of PIP3 gen-erated by wild-type BMNs in response to either fMLP or C5a were not sta-tistically significantly affected by inhibition of p110bwith 40 nMTGX221,nor were they substantially reduced in b-KO neutrophils (Fig. 3, C and D).However, TGX221 reduced the mean amount of PIP3 generated in g-KOcells in response to either fMLP or C5a by ~5 to 10%, which suggests that

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    p110b might make a very small contribution to basal or GPCR-stimulatedPIP3 formation. Consistent with the lack of a major role for p110b in thePIP3 responses to C5a and fMLP, the PI3K-dependent formation of ROS inresponse to these agonistswas also unaffected by 40 nMTGX221 (fig. S5A).

    Both intact Gbg- and Rac/Cdc42-binding domains arerequired to support p110b-dependent neutrophilactivation by FcgRs and b2 integrinsWe previously described an essential role for p110b in the responses ofmouse neutrophils to immune complexes and adhesive surfaces (31). Theseligands are recognized initially by FcgRs and integrins, respectively, whichthen drive a highly cooperative and complex sequence of “outside-in” and“inside-out” signaling events that regulate increased binding avidity,spreading, granule secretion, and NADPH (reduced form of nicotinamideadenine dinucleotide phosphate) oxidase (NOX)–dependent formation ofROS (43, 44). Furthermore, other observations suggest that these two re-ceptor systems share several common signaling elements, including phospho-rylation of the g chain of FcRs (Fc receptors) by an Src family kinase and theconsequent recruitment and activation of Syk (45).

    Our previous studies suggested that a paracrine loop involving the in-flammatorymediator leukotrieneB4 (LTB4) and its GPCRBLT1 is requireddownstream of FcgRs for the efficient activation of p110b and NOX (31).Therefore, we were interested in exploring the potential involvement of thedirect binding of Gbg to p110b in this response. The addition of wild-typeBMNs to immobilized immunoglobulin G (IgG)–bovine serum albumin(BSA) induced cell adhesion, spreading, and the production of ROS, whichwas maximal at around 10 min and was maintained for over 40 min (Fig. 4,A to E). Pharmacological (40 nMTGX221) or genetic (b-KO) inhibition ofp110b reduced each of these responses by about 70% (Fig. 4, C to E),confirming a central role for p110b. Cell adhesion and the production ofROS, and to a lesser extent cell spreading, were substantially reduced inb-Gbg BMNs compared to those in wild-type BMNs (Fig. 4, C to E). Sub-stantial reductions in adhesion and ROS formation were also seen withb-RBDBMNs (Fig. 4, C toE). The addition of 40 nMTGX221 still resultedin small reductions in the ROS responses of b-Gbg and b-RBD neutrophils(Fig. 4E), which suggests that preventing the direct binding of Gbg, Rac, orCdc42 to p110b results in the substantial, but incomplete, elimination of therole of p110b in this response.

    We extended these observations by evaluating the role of p110b in thegeneration of ROS in response to the FcgR-dependent phagocytosis of IgG-opsonized sheep red blood cells (SRBCs) by BMNs primed with tumor ne-crosis factor–a (TNF-a) and granulocyte M-CSF (GM-CSF) [nonprimedcells do not generate ROS in response to IgG-SRBCs (46)]. The additionof wild-type BMNs to IgG-SRBCs elicited the generation of less but still asubstantial amount of ROS than did their addition to IgG-BSA, which wasmaximal at around 7min and lasted for more than 40min (Fig. 4B). Similarto the IgG-BSA response described earlier, production of ROS in responseto the phagocytosis of IgG-SRBCswas highly dependent on p110b activity,being reduced by 70% in b-KOBMNs or by the addition of TGX221 towild-type BMNs (fig. S5B). The amounts of ROS produced in response to IgG-SRBCswere also greatly reduced in b-Gbg or b-RBD neutrophils (fig. S5B),with 40 nM TGX221 reducing the response further only in b-Gbg cells,which suggests that the RBD plays a particularly important role in the reg-ulation of p110b in this context.

    Neutrophils also adhere, spread, and generate ROS in response to theengagement of b2 integrins with extracellular ligands (47), although theROS response in our experiments did not require BLT1 (fig. S6). Adhesionof BMNs to the physiologicalMac1 ligand fibrinogen (FGN) in the presenceof the proinflammatory cytokine TNF-a (Fig. 4C) or onto the synthetic mul-tivalent integrin ligand poly–Arg-Gly-Asp+ (poly-RGD+) (Fig. 4C) was

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    Fig. 2. SynergisticPIP3 production byBMDMs in response to costimulationofRTKs and GPCRs is dependent on p110b and requires binding to Gbg pro-teins. (A) Left: BMDMs (1.2 × 106 cells) from WT mice were serum-starvedovernight beforebeingstimulated for 30 swith 30nMC5aorM-CSF (30ng/ml)alone or in combination, as indicated. PIP3wasquantifiedbyMSasdescribedfor Fig. 1.Dataaremeans±SEMofat least 10experiments, eachperformed induplicate, and are expressed as a percentage of the response to M-CSF.Right: Ratios for costimulation (the PIP3 response to simultaneous additionof C5a and M-CSF) divided by additive (the sum of the individual PIP3 re-sponses to C5a and M-CSF) for the same experiments were calculated asdescribed in Materials and Methods. (B) Left: WT (closed bars), b-KO (openbars), d-KI (dark gray bars), and g-KO (light gray bars) BMDMs (1.2 × 106)were left unstimulated (control) or were stimulated with the indicated agonists,

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    andPIP3/PI ratioswerequantitatedasdescribed in (A).Dataaremeans±SEMof at least three experiments, eachperformed in duplicate, and are expressedasapercentageof thePIP3abundanceofWTmacrophages in response toM-CSF. Data for the unstimulated and singly stimulated cells include data fromtheexperiments shown in (A)andFig. 1 (BandD).Right:Ratios (costimulation/additive) for the sameexperimentswerecalculatedasdescribed in (A). (C) Left:BMDMs from WT (closed bars), b-RBD (hatched bars), b-Gbg (striped bars),andb-KO (openbars)micewerepreparedand treatedasdescribed in (A).Dataaremeans±SEMof at least three experiments, eachperformed in duplicate, andareexpressedasapercentageof theresponseofWTcells toM-CSF.Right:Ratios(costimulation/additive) for the sameexperimentswerecalculatedasdescribed in(A). *P

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    largely unaffected by the inhibition of p110b. However, cell spreading andthe production of ROS in response to both stimuli were substantially reducedin either b-KO BMNs or TGX221-treated wild-type BMNs (Fig. 4, D andE). Furthermore, ROS production and, to a lesser extent, cell spreading werereduced in b-Gbg BMNs compared to those in wild-type BMNs (Fig. 4, Dand E), whereas b-RBD BMNs displayed reduced ROS production but hadunaffected spreading (Fig. 4, D and E). As before, preincubationwith 40 nMTGX221 elicited further small reductions in ROS generation by b-Gbg andb-RBD BMNs; however, this did not reach statistical significance (Fig. 4E).

    Together, these data suggest that both an intact RBD and the directbinding of Gbg subunits substantially contribute to the regulation of p110bin FcgR- and b2 integrin–mediated signaling pathways in neutrophils. Theextent to which p110b activity controls various downstream elements, suchas receptor clustering and activation, cytoskeletal rearrangement, or oxidaseactivation, is difficult to discern; however, because the relationships betweenthese pathways are still incompletely understood. From our data, it appearsthat adhesion, spreading, and ROS formation in response to IgG-BSA aresimilarly dependent on p110b; however, ROS formation in response to in-tegrin engagement is much more dependent on p110b than is cell adhesion.Ideally,wewould havewished tomeasure PIP3 formation in response to theseagonists as amore direct readout of p110b activity. Unfortunately, the sizes ofthe PIP3 responses elicited in response to TNF-a–FGN, IgG-BSA, or IgG-SRBCs were too small to measure with enough precision to enable a useful

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    dissection of potential regulatory inputsinto p110b. This is possibly because ofthe imprecise time at which the neutro-phils first encountered the stimuli inthese types of assay. However, a statisti-cally significant 50% increase in PIP3abundance above that in unstimulatedcells was measured 10 min after BMNswere plated onto poly-RGD+ (Fig. 5),and this was similarly reduced in b-KO,b-Gbg, and b-RBD BMNs or upon theaddition of 40 nM TGX221 towild-typeBMNs (Fig. 5). These data suggest thatthe RBD and Gbg binding are requiredfor p110b stimulation in response to in-tegrin activation. Furthermore, the basalamounts of PIP3 inBMNs in suspensionwere reduced further by the addition of thepan-PI3K inhibitorwortmannin (100nM),which suggests that they were generatedthrougha largelyp110b-independent route(Fig. 5).

    DISCUSSION

    There is steadily accumulating evidenceof selective roles for p110b in individualcell responses and animal physiology(1, 3, 32). p110b is also implicated indriving pathology, such as thrombosis(48), lung fibrosis (9), autoimmune in-flammation (18, 39), and cancer (49).Thus, p110b has been suggested to bea potentially useful therapeutic target(31, 50, 51). There has been substantialprogress in defining some of the molec-ular properties of p85-p110b dimers that

    may enable p110b to play such selective roles (24, 30, 52), but the extent towhich they are individually or cooperatively responsible in a given contextin vivo remains uncertain. In particular, there is debate over the extent towhich p110b is a direct transducer of GPCR signals. p85-p110b heterodi-mers were initially characterized as responding to direct activation by Gbgsubunits in vitro, though the extent of this activation was modest in theabsence of costimulation by tyrosine-phosphorylated peptides (24, 25).Furthermore, pharmacological or genetic inhibition of p110b suggested thatp85-p110b plays a widespread role downstream of several GPCRs (for exam-ple, receptors for lysophosphatidic acid, CXCL12, or sphingosine 1-phosphate)and acts redundantly with p101-p110g heterodimers in cells in which bothheterodimers are present (for example, cells of hematopoietic origin) (27, 28).In contrast, p110b is generally accepted to play a confusingly minor roledownstream of many RTKs, particularly those responding to growth factors,such as PDGF, EGF, or insulin (27, 28, 53), the exception being in circum-stances in which p110a activity is low, for example, after p110a-selectivepharmacological inhibition (54, 55) or when p110a signaling is inhibitedby aPTEN-PIP3–mediated negative feedback loop (56). Furthermore, a studydescribed an alternative, indirect route for the Gbg-dependent activation ofp110b through theGbg-dependent regulationof guanine nucleotide exchangeon Rac and the subsequent binding of GTP-Rac to the RBD of p110b (9).

    The identification of the Gbg-binding site on p110b enabled the gener-ation of point mutants (KK532/533DD) that selectively ablate the binding

    Fig. 3. PIP3 generation inBMNs in response to theGPCRagonists fMLPandC5a requires p110g but not p110b.(A and B) WT BMNs (0.5 × 106) were stimulated in suspension with 10 mM fMLP (A) or 100 nM C5a (B) for theindicated times. The reactionswere quenched, and lipidswere extracted, derivatized, and quantitated byMSasdescribed earlier. Data are means ± SEM of the PIP3/PI ratio of at least three experiments, each performed in atleast duplicate. (C and D) BMNs (0.5 × 106) from WT, b-KO, and g-KO mice were preincubated with 40 nMTGX221 (hatched bars) or 0.05% DMSO (vehicle, closed bars) before being stimulated for 10 s with fMLP (C)or C5a (D). Reactions were then stopped, and lipids were extracted and quantitated as described earlier. ThePIP3/PI ratio in unstimulatedWT BMNs (WT-CON) is also shown. Data are means ± SEM of the PIP3/PI ratio of atleast three experiments, each performed in at least duplicate. NS, not significant. ***P < 0.005 by one-wayANOVA with Holm-Sidak post hoc test comparisons as indicated (C and D).

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    Fig. 4. Adhesion, spreading, and ROS production in BMNs in response to (CandD) BMNs from the indicatedmicewerepretreated for 10minwith either

    immune complexes and adhesive proteins are p110b-dependent responses.(A) BMNs fromWT, b-Gbg, b-RBD, and b-KOmicewere incubated for 10minwith either DMSOor 40 nMTGX221 before being applied to glass coverslipscoatedwithpoly-RGD+.Cellswere incubatedat 37°C for 20min, andadherentcells were fixed in 3.7% paraformaldehyde, washed, and mounted on glassmicroslides before being visualized with a Nikon Ti-E Live Cell Imager invertedmicroscopewitha20×objective lens. Imagesof a1:15 fieldof viewof adherentspread (S) and nonspread (NS) cells are representative of three experimentsperformed induplicate (except for cells from b-KOmice, forwhichn=2). (B)WTBMNs (0.5 × 106) were preincubated with horseradish peroxidase (HRP) andluminol before being added to a 96-well plate coated with IgG-BSA (triangles),FGN(diamonds),orpoly-RGD+(circles),or toaplatecontaining IgG-opsonizedSRBCs (squares).ROSgenerationwasmeasured in triplicate foreachconditionby chemiluminescence and recorded with a Berthold MicroLumat Plus lumi-nometer and are expressed as relative light units (RLUs) per second. Dataare fromoneexperiment andare representativeof at least threeexperiments.

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    DMSO or 40 nM TGX221 before being incubated on duplicate coverslipscoatedwith IgG-BSA, FGN, or poly-RGD+, as indicated, andwere processedas described for (A). Images were analyzed for cell adhesion and spreadingwith ImageJ software (Fiji), selecting for size and brightness as described inMaterials and Methods. Data are means ± SEM of the number of adherentcells per field of view (C) and of the percentage of spread cells (D) from threeexperiments performed in duplicate (except for cells from b-KOmice, for whichn = 2). (E) BMNs (0.5 × 106) from WT, b-RBD, b-Gbg, and b-KO mice werepreincubated with HRP/luminol in the presence of 40 nM TGX221 (hatchedbars) or DMSO (0.05%, closed bars) before being added to wells precoatedwith IgG-BSA, FGN, or poly-RGD+, as indicated. ROS was then measured asdescribedearlier.Dataaremeans±SEM foraccumulated light emission (RLU)over a 20-min recording of at least three experiments performed in at leastduplicate and are expressed as a percentage of the ROS generated inDMSO-treated WT BMNs. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P <0.001 by t test with Holm-Sidak corrections for multiple comparisons.

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    of Gbg but have no effect on basal p110b activity, its interaction with Rab5,or the activation of p85-p110b heterodimers by phosphotyrosine peptides(24). This enabled us to generate a knock-in mouse (b-Gbg) expressing amutant p110b to which Gbg binding was selectively blocked, which thenprovided us with an opportunity to investigate the role of this interactionin vivo. The abundance of p110b-KK526/527DDwas normal (comparableto that of the protein in wild-type cells) in primary macrophages and neu-trophils, and homozygous knock-in mice were born at normal Mendelianratios with no overt phenotype. This contrasts with the growth and develop-mental defects seen in p110b KO or p110b kinase–inactive knock-in mice,which indicates that the b-Gbg mice retain some p110b function (26–28).

    We chose to assess the effect of the KK526/527DD mutation on theGPCR-stimulated formation of PIP3 in macrophages and neutrophils,because these cells contain all four class I PI3K isoforms and exhibitboth GPCR and RTK-dependent PI3K signaling. We measured the ac-cumulation of PIP3 formation at early times as the most linear readout ofclass I PI3K activity available. Individual stimulation of BMDMs by C5aor of BMNs by C5a or fMLP resulted in the rapid synthesis of PIP3 in amanner that was almost entirely dependent on p110g and largely independentof p110b. Previous work suggested that the C5a-stimulated phosphorylationof Akt in macrophages is substantially dependent on both p110b and p110g(28). At present, we have no explanation for the difference between theseresults and ours; however, the primary role of p110g downstream of GPCRsignaling in neutrophils agrees with most previous studies and concurs withthe much more sensitive and potent Gbg-dependent activation of p101-p110g than of p85-p110b in vitro (24, 57, 58).

    In BMDMs, the generation of PIP3 in response to M-CSF enabled a di-rect comparison of the involvement of different PI3K isoforms in similarlysized RTK and GPCR responses. TheM-CSF–stimulated generation of PIP3at 30 s involved cooperation between all three class Ia PI3Ks, with similarcontributions from p110a, p110b, and p110d. In principle, the widely held

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    assumption that class Ia p110 isoforms share a commonpool of phosphotyrosine-binding regulatory subunits provides a natural mechanism for redundancydownstream of RTKs, although previous work implied a predominant role foreither p110a or p110d in the responses of macrophages to M-CSF (59–61). Itseems likely that the explanation for these apparently disparate results lies in thedifferent assays, time scales, and cell preparations used. It is also possible that thelevel of redundancyamongclass Ia isoformshasbeengenerally underestimated inthe past because of the use of cellular preparations exhibiting widely differing re-lative amountsofp110 isoforms,poorquantitationof class IPI3Kactivity, or both.

    Coincident stimulation of BMDMs by C5a and M-CSF resulted in thesynergistic productionofPIP3 in amounts thatwere about twice those thatwereexpected on the basis of a purely additive effect. Furthermore, loss of p110bselectively removed this synergistic component of the response, whereas in-hibition of p110a, p110g, or p110d had no effect on the degree of synergy. Inaddition, b-Gbg macrophages also failed to support the synergistic produc-tion of PIP3 in response to C5a andM-CSF, which suggests that this role forp110b is entirely dependent on its direct interactionwithGbg. The role of theRBD in regulating p110b activity in these responseswas less clear; themeanamount of PIP3 produced by b-RBDBMNs in response to both C5a andM-CSFwas ~20% less than that produced by wild-type BMNs, but this did notreach statistical significance.

    These results suggest that PI3Kb does indeed have a direct role in trans-ducing GPCR signals but only in the context of parallel activation of anRTK. Thus, Gbg binding to a p85-p110b heterodimer alone is insufficientto elicit substantial PIP3 production; however, in the context of simultaneousp85-phosphotyrosine docking (as occurs during parallel RTK activation),the binding ofGbg to p110b supports additional PI3Kb activation, giving riseto a synergistic increase in PIP3 formation. These conclusions align closelywith studies describing the direct and synergistic activation of p85-p110bheterodimers by Gbg subunits and phosphotyrosine peptides in vitro, andalso our current mechanistic understanding of howGbg and phosphotyrosinedocking might cause enzyme activation (by increasing membrane dwell timeand allosteric activation, respectively) (6, 24, 49).

    We sought to extend our understanding of the role of Gbg in the regula-tion of p110b by investigating ROS production in BMNs in response toseveral stimuli. We and others previously established that the stimulationof mouse neutrophils with C5a and fMLP results in the rapid and transientproduction of ROS, which is dependent on p110g (38). We also described arole for p110b in the more slowly developing and sustained production ofROS in response to FcgR-mediated recognition of immune complexes andb2 integrin–mediated adhesion (31). Here, we confirmed that BMN ROSresponses to immobilized IgG-BSA or adhesion to FGN or poly-RGD+are all dependent on p110b activity. Furthermore, we extended our studiesto demonstrate that ROS generation in response to the FcgR-mediatedphagocytosis of IgG-opsonized SRBCs was also highly dependent onp110b, implicating p110b activity in an important role downstream of theactivation of FcgR and supporting previous observations that proposed acommon mechanism used by both FcgR and integrins to activate NOX2in neutrophils. ROS responses to IgG-BSA, adhesion, or IgG-SRBCs werealso substantially reduced in b-Gbg and b-RBD BMNs compared to thosein wild-type BMNs. The relative scale of these reductions compared tothose seen in b-KO cells and the extent of additional TGX221-dependentinhibition suggest that both direct Gbg binding to p110b and input throughthe RBD play important and cooperative roles in p110b regulation. Theinput through the RBD is most important in ROS production in responseto IgG-SRBCs and poly-RGD+, and this may correlate with the central rolethat GTP-Rac plays in these responses (62).

    These results indicate that Gbg subunits are involved in the activation ofp110b downstream of FcgRs and b2 integrins, which directly implicatesGPCRs in these responses. BLT1, the GPCR for LTB4, has been suggested

    Fig. 5. The p110b-dependent generation of PIP3 in BMNs in response to ad-hesion to poly-RGD+ requires both an intact RBD and binding to Gbg pro-teins. BMNs fromWT, b-RBD, b-Gbg, and b-KOmicewerepreincubatedwith40 nM TGX221 or vehicle control before being added to glass coverslipscoated with poly-RGD+ or were left in suspension. Where indicated, WTBMNs were additionally preincubated with 100 nM wortmannin (wort). Cellswere allowed to adhereand spread for 10minat 37°Cand thenharvested forPIP3 measurement by MS as described earlier. Data are means ± SEM ofPIP3/PI ratios from threeexperiments (except for data fromwortmannin-treatedcells, for whichn=2), eachperformed in duplicate.N.D., not determined. *P<0.05byone-sample t testwithHolm-Sidakcorrection formultiplecomparisons.

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    to play a role in FcgR-mediated phagocytosis by macrophages (63) and inROS production by neutrophils in response to immobilized immunecomplexes (31). Neutrophils can generate substantial amounts of LTB4in response to FcgR activation, and thus, there is the potential to set up aparacrine loop in vitro that involves coincident signaling through GPCRand tyrosine kinase signaling; FcgRs initiate signaling through non-RTKsof the Src and Syk families (64). However, BLT1 did not appear to beinvolved in the generation of ROS in response to b2 integrin stimulation(fig. S6), which suggests that GPCR signaling plays a previously unchar-acterized role in these responses.

    Our results suggest that the direct binding of Gbg subunits to p110b co-operates with the binding of Rac and Cdc42 to the p110b-RBD and also thebinding of the p85SH2domain to phosphotyrosines to enable p85-p110b toplay a unique role downstream of coincident GPCR and RTK signaling.However, the extent towhich the interaction with Gbg can operate indepen-dently of the other regulatory inputs to transduce isolatedGPCR signals (forexample, in the absence of the more dominant p101-p110g isoform) is stilldifficult to assess. No mutations in p110b have yet been defined that selec-tively ablate allosteric activation through SH2-phosphotyrosine docking,and almost all cells in their “basal” state probably receive survival signalsthrough low-level, phosphotyrosine-mediated activation of class I PI3Ks, forexample, through integrin-mediated adhesion (65). Nevertheless, with orwithout coincident RTK signaling, there is a clear case for searching for pre-viously uncharacterized GPCR inputs where p110b plays a major role inphysiology or pathology.

    MATERIALS AND METHODS

    ReagentsBovineFGN, poly-RGD+, fMLP, luminol, TMS-diazomethane,wortmannin,fatty acid–free BSA, and HRP were from Sigma-Aldrich. Murine GM-CSFandM-CSF were from PeproTech, whereas murine TNF-a and recombinanthuman C5a were from R&D Systems. SRBCs in Alsever’s were from TCSBiosciences. Hanks’ balanced salt solution and Dulbecco’s phosphate-buffered saline (dPBS) with Ca2+ and Mg2+ were from Sigma. The isoform-selective PI3K inhibitors IC87114 and TGX221 were from Calbiochem,whereasBYL719was fromActiveBiochem.All tissue culture productswerefrom Invitrogen. All buffer components were from Sigma-Aldrich and wereendotoxin-free or low-endotoxin, as available. Internal standards for lipidanalysis, 1-heptadecanoyl-2-hexadecanoyl-sn-glycero-3-(phosphoinositol3,4,5-trisphosphate) (C17:0/C16:0-PIP3, as a hepta sodium salt), andC17:0/C16:0-PI were synthesized at the Babraham Institute. All chemicalsand solutions were of analytical reagent grade.

    Mouse strainsMice lacking p110g (g-KO) (33) or p110b (b-KO) (31) andmice expressinga kinase-inactive p110d (d-KI, p110d-D910A) (34) or point mutations inthe RBD of p110b (b-RBD; Jackson Laboratory, JAX strain 025930) (9)have been previously described. We generated knock-in mice expressinga point-mutated PI3Kb that is deficient in binding to Gbg dimers (describedbelow and in the Supplementary Materials). In all experiments, mice werecompared with appropriate age- and strain-matched wild-type controls.Mice were housed in the Biological Support Unit at the Babraham Instituteunder specific pathogen–free conditions. All work was performed underHome Office Project license PPL 70/8100.

    Generation of knock-in mice expressingGbg-insensitive p110bThe amino acid residues that bind to and enable activation of p110b byGbgdimers, K532 and K533, have previously been identified in the human

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    sequence (24). Through sequence alignment of human and mouse p110b,we identified K526 and K527 as the corresponding residues in mouse p110b(fig. S1A). The Pik3cbKK526,527DD targeting construct was generated with arecombineering-based methodology as previously described (66). Thismethod uses the homologous recombination functions of the l phage Redgenes to subclone and retrieveDNAfrombacterial artificial chromosomes intohigher-copy plasmids and enables insertions, mutations, or deletions to bemade in the retrieved gene of interest. For full details about generation ofthe targeting construct and resultant knock-inmice expressingGbg-insensitivep110b, see the Supplementary Materials.

    Preparation and stimulation of BMDMsBMDMs were prepared essentially as previously described (60). Briefly,flushed bone marrow was resuspended in complete medium [RPMI 1640medium containing L-glutamine supplemented with 10% heat-inactivatedfetal bovine serum (HI-FBS), 1%penicillin and streptomycin, 1mMsodiumpyruvate, 1% nonessential amino acids, 0.5 mM b-mercaptoethanol, and M-CSF (20 ng/ml)]. Cells were distributed to 10-cm2 tissue culture plates (10 ×106 cells per plate) and cultured for 72 hours at 37°C, 5% CO2. BMDMs(nonadherent cells) were harvested and stored frozen in 90% HI-FBS and10% DMSO under liquid nitrogen until use. Before experiments were per-formed, BMDMswere thawed and grown in completemedium on nontissueculture dishes (1.2 × 106 cells per dish) for a further 3 days before detaching(with versene) and replating on 10-cm2 tissue culture dishes (at 1.2 × 106 cellsper dish). Flow cytometric analysis of cells (to detect CD11b and F4/80) re-vealed that >95% of the cells were macrophages. Before stimulation, theBMDMs were starved overnight in medium in the absence of HI-FBS andM-CSF. Where indicated in the figure legends, cells were pretreated with in-hibitors (for 10 min at 37°C, 5% CO2) before being stimulated for the timesindicated in the figure legends (at 37°C, 5%CO2). Reactionswere terminatedby aspiration of the buffer and the addition of 1 ml of ice-cold 1MHCl. Ad-herent cells, on ice, were harvested by scraping and transferred to 2-ml safe-lock Eppendorf tubes on ice for centrifugation at 15,000g at 4°C and removalof the supernatant. At this point, cell pellets could be snap-frozen in liquidnitrogen and stored at −80°C before proceeding with lipid extraction.

    Preparation of BMNsForROS assays and soluble agonist lipid analysis, maturemouse neutrophilswere isolated from bone marrow at room temperature on a discontinuousPercoll+ gradient as described previously (38). Purity was determined by cy-tospin and Reastain QUICK-DIFF (Reagena) staining, and preparationswere at least 70 to 85% pure. After washing, BMNs were resuspended indPBS with Ca2+ and Mg2+, glucose (1 g/liter), and 4 mM sodium bi-carbonate (dPBS+). For phospholipid analysis of adherent cells, BMNswere prepared at 4°C as described previously (67). Cells (60 to 80% pure,as assessed by cytospin) were resuspended at 5 × 106 cells/ml in dPBS+ inthe presence of 0.05%endotoxin and essentially fatty acid–freeBSA (dPBS++).Cell suspensions were allowed to equilibrate for 5 min at room temperatureand then for 10 min at 37°C in the presence or absence of 40 nM TGX221,100nMwortmannin, orDMSO(vehicle control), as indicated, before adhesion.

    Preparation of adhesive surfacesImmune complexes were immobilized by coating luminometer plate wells(Berthold Technologies; for ROS assays) or glass coverslips (for adhesionassays) overnight at 4°Cwith PBS containing endotoxin-free and essentiallyfatty acid–free BSA (100 mg/ml). After washing in PBS, wells or coverslipswere blocked with 1% fat-free milk in PBS for 1 hour at room temperature.After further washing, immune complexes were formed by the addition of a1:10,000 dilution ofmonoclonal anti-BSAantibody (Sigma,B2901) in dPBS+for 1 hour at room temperature, followed by washing to remove unbound

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    antibodies. Bovine FGN (150 mg/ml) was adsorbed onto luminometer wells(for ROS assays) or coverslips (for adhesion assays) overnight at 4°C, andsurfaces were washed with dPBS before assays were performed. BMNswere preincubated with murine TNF-a (20 ng/ml) for 10 min at 37°C inthe absence or presence of inhibitor before being added to FGN-coatedsurfaces. Poly-RGD+ in dPBS+ was adsorbed onto luminometer plates(at 20 mg/ml for ROS assays) or glass coverslips (at 20 mg/ml for adhesionassays or at 100 mg/ml for phospholipid analysis) for 3 hours at room tem-perature and were washed with dPBS+ before the BMNs were added.

    Preparation of IgG-opsonized SRBCsIgG-opsonized SRBCs were prepared for ROS assays as previously de-scribed (46). Briefly, 10 ml of SRBCs in Alsever’s (TCS Biosciences) waswashed, centrifuged at 1500g for 4min, and resuspended in 1ml of dPBS++.SRBCswere opsonizedwith a 1:1000 dilution of IgG anti-SRBC (rabbit,MPBiomedicals), rotating end over end at room temperature for 20 min. SRBCswere then washed twice and resuspended in 800 ml of dPBS++.

    Measurement of ROS productionRate kinetics of ROS production were measured by chemiluminescencewith a luminol-based assay in polystyrene 96-well plates (Berthold Technol-ogies) as described previously (68). Cells (0.5 × 106) were incubated with150 mM luminol and HRP (18.75 U/ml) for 10 min at 37°C in the presenceor absence of 40 nM TGX221 (or DMSO vehicle control, 0.05%) as indi-cated in the figure legends. BMNs were added manually to wells precoatedwith FGN (150 mg/ml), poly-RGD+ (20 mg/ml), or immobilized immunecomplexes (1:10,000 anti-BSA), which were prepared as described earlier.For IgG-opsonized SRBC-generated ROS assays, BMNs were primed for1 hour at 37°C in the presence of mouse TNF-a (1000 U/ml) and GM-CSF(100 ng/ml). For all assays, measurements were started immediately, andlight emission was recorded by a Berthold MicroLumat Plus luminometer(Berthold Technologies). Data output was in RLUs per second or totalRLUs integrated over the indicated measured time periods.

    BMN adhesion and spreading assaysBMNs (0.5 × 106 cells for poly-RGD+ and IgG-BSA; 1 × 106 cells forFGN) from wild-type, p110b Gbg–insensitive (b-Gbg), p110b RBD–insensitive (b-RBD), or p110b knockout (b-KO) mice were applied induplicate to 15-mm glass coverslips in 24-well tissue culture plates pre-coated with poly-RGD+ (20 mg/ml), IgG-BSA (1:10,000 anti-BSA), orFGN (150 mg/ml), as described earlier, after a 10-min pretreatment at 37°Cwith 40 nM TGX221 or DMSO vehicle. Cells were incubated at 37°C for20 min, nonadherent cells were aspirated, and adherent cells were fixed in3.7% paraformaldehyde in dPBS (pH 7.4) for 15 min at room temperature.After being washed three times in dPBS, the coverslips were rinsed inMilliQ double-distilledwater before beingmounted on glassmicroslideswithAqua-Poly/Mount antifading solution (Polysciences). Cells were visualizedon wide field by differential interference contrast, and images were capturedwith a Nikon Ti-E Live Cell Imager inverted microscopewith an integratedcamera, using a 20× objective lens. Images were analyzed for cell adhesionand spreading in FijiX64 for 10 to 15 (poly-RGD+), 15 to 20 (IgG-BSA), or25 to 30 (FGN) random fields of view per coverslip, selecting for cell sizeand cell brightness.

    BMN adhesion assays: Preparation for lipid analysisIsolated BMNs (1 × 106 cells in 200 ml) were added at 37°C to 32-mmglasscoverslips within 35-mm culture dishes precoated with poly-RGD+, as de-scribed earlier, containing 800 ml of dPBS++ (in the presence of 40 nMTGX221, 100 nM wortmannin, or 0.05% DMSO as required) and wereallowed to adhere for 10min at 37°C.Reactionswere terminatedby aspirating

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    the buffer, adding 0.5ml of ice-cold 1MHCl, and placing the dishes on ice.Adherent cells were scraped from coverslips and transferred to 2-ml safe-lock Eppendorf tubes on ice. For suspension controls, 1 × 106 BMNs wereincubated at 37°C for 10min in the absence or presence of 40 nMTGX221,100 nMwortmannin, or 0.05%DMSO as required. Reactionswere stoppedby the addition of 1.5 ml of ice-cold 1MHCl, and the tubes were placed onice. Cells from adhesion and suspension samples were pelleted by centrifuga-tion (18,000g for 10 min at 4°C), and the supernatant was removed. Cellspellets were then processed for lipid analysis.

    Lipid analysisBMN cell pellets from adhesion assays or BMDM cell pellets prepared asdescribed earlier were resuspended in 920 ml of primary extraction solution[CHCl3/MeOH/1 M HCl (484/242/23.22)/H2O, 750:170 (v/v)]. The inter-nal standards C17:0/C16:0-PI (100 ng) and C17:0/C16:0-PIP3 (10 ng) wereadded to the initial extract resuspensions. The lipids were extracted and de-rivatized with trimethylsilyl-diazomethane, and PIP3 and PI were analyzedby MS, using neutral loss of derivatized head groups with an AB SciexQTRAP 4000 connected to a Waters Acquity UPLC system as describedby Clark et al. (69), with the exception that final samples were dried in arotary SpeedVac evaporator rather than under nitrogen. For lipid measure-ments inBMNs exposed to soluble agonists, 0.5 × 106BMNs in 150 mlwereequilibrated to 37°C for 3min before stimulation at 37°Cwith 10 mM fMLPor 100 nM C5a for the times indicated in the figure legends. Where indi-cated in the figure legends, BMNs were preincubated for 10 min at 37°Cwith 40 nM TGX221 or 0.05% DMSO before being stimulated. Reactionswere terminated by the addition of 750 ml of ice-cold CHCl3/MeOH/1 MHCl (484/242/23.22), and samples were placed on ice until extraction (whichoccurred within 10 min). C17:0/C16:0-PIP3 and C17:0/C16:0-PI internalstandards were added, and the samples were processed as described earlier.Dataweregenerated as response ratios, calculated bynormalizing themultiplereaction monitoring–targeted, lipid-integrated response area to that of aknown amount of relevant internal standard. Where indicated, PIP3 amountswere divided by PI amounts to correct for any cell input variability betweenexperiments and are presented as nominal PIP3/PI ratios. For the BMDMPIP3 data presented in Fig. 2, for each separate experiment, the “additive re-sponse”was calculated by adding the individual C5a-mediated PIP3 responseabove control (control being the amount of PIP3 in unstimulated cells) to theindividual M-CSF–mediated response above control, whereas the “costimu-lation response” represents the actual response to both agonists added simul-taneously (C5a andM-CSF) above control (that is, a single subtraction for theamount of PIP3 in unstimulated cellswas performed).A ratio of costimulationresponse dividedby the additive responsewas then calculated for each separateexperiment, and the collected data were subjected to statistical analysis.

    StatisticsData are means ± SEM of at least three experiments performed with at leastduplicate samples (unless otherwise indicated). BMNs and BMDMs wereisolated from at least two pooled mice of each genotype for each experi-ment. One-way ANOVA followed by Holm-Sidak post hoc tests were per-formed on all BMN data (PIP3 and ROS) and for BMDM PIP3 responses,except for the use of ratio paired t tests with Holm-Sidak post hoc test tocompare costimulation and additive PIP3 responses to C5a andM-CSF. Foreach test, *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.0001.

    SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/9/441/ra82/DC1MethodsFig. S1. Targeting of Pik3cbKK526,527DD.Fig. S2. Confirmation of the correct targeting of Pik3cbKK526,527DD.

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    Fig. S3. p110b quantities in BMDMs and BMNs are unaffected by the introduction of theRBD- and Gbg-insensitive mutations.Fig. S4. Dose responses of GPCR (C5a) and RTK (M-CSF) agonists on PIP3 productionby BMDMs.Fig. S5. ROS generation in BMNs in response to the phagocytosis of IgG-SRBCs, but notsoluble GPCR agonists, requires p110b activity.Fig. S6. ROS generation in BMNs in response to adhesion to poly-RGD+, but not immunecomplexes, is independent of BLT1 activation.Reference (70)

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    Acknowledgments:Wewould like to thankmembersof theSmall AnimalBreedingUnit at theBabraham Institute for animal husbandry; R.Williams (Medical ResearchCouncil Laboratory ofMolecular Biology, Cambridge) for communicating essential information about the direct inter-action between Gbg and p110b; J. Clark for help with the MS analysis of lipids; A. Segonds-Pichon for statistical analysis; S. Walker and H. Okkenhaug (Babraham Institute ImagingFacility) for assistance with image analysis; and S. Suire and D. Gyori for helpful discussions.Funding: This work was supported by funding from the Biotechnology andBiological SciencesResearch Council (BBSRC) UK (BB/J004456/1 to K.E.A., L.R.S., and P.T.H.), the WelcomeTrust (WT085889MA to T.C. and S.K.), the NIH (GM112524 to J.M.B.), and the European Re-search Council (RASTARGET to J.D.). D.M.H. is a recipient of a BBSRC PhD studentship.Author contributions: K.E.A., D.M.H., S.K., and T.C. performed experiments; K.E.A., D.H.,P.T.H., and L.R.S. analyzed data; R.F., J.D., and J.M.B. supplied important resources andinformation; P.T.H., K.E.A., and L.S. designed research; and P.T.H., K.E.A., T.C., and L.R.S.wrote the article. Competing interests: The authors declare that they have no competing in-terests. Data and materials availability: Use of the p110b KO and p110d D910A-KI mice re-quires a material transfer agreement from the Ludwig Institute for Cancer Research.

    Submitted 9 December 2015Accepted 29 July 2016Final Publication 16 August 201610.1126/scisignal.aae0453Citation: D. M. Houslay, K. E. Anderson, T. Chessa, S. Kulkarni, R. Fritsch, J. Downward,J. M. Backer, L. R. Stephens, P. T. Hawkins, Coincident signals from GPCRs and receptortyrosine kinases are uniquely transduced by PI3Kb in myeloid cells. Sci. Signal. 9, ra82(2016).

    .SCIENCESIGNALING.org 16 August 2016 Vol 9 Issue 441 ra82 12

    http://stke.sciencemag.org/

  • in myeloid cellsβPI3KCoincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by

    Backer, Len R. Stephens and Phillip T. HawkinsDaniel M. Houslay, Karen E. Anderson, Tamara Chessa, Suhasini Kulkarni, Ralph Fritsch, Julian Downward, Jonathan M.

    DOI: 10.1126/scisignal.aae0453 (441), ra82.9Sci. Signal.

    to activate G proteins.βdepended on the ability of PI3Ksimultaneous stimulation of both receptor types. Furthermore, effective responses of neutrophils to inflammatory stimuli

    concentration in response to3 uniquely induced synergistic increases in PIPβcombination. Of all the PI3K isoforms, PI3K responses of macrophages and neutrophils to RTK and GPCR agonists individually or in3measured the PIP

    unable to transduce GPCR signals andβ. generated knock-in mice expressing a mutant PI3Ket altypes. Houslay transduces signals from both receptorβtyrosine kinases (RTKs) and one isoform mediates signals from GPCRs, PI3K

    in response to stimulation. Whereas some PI3K isoforms mediate signaling from receptor3second messenger PIPPhosphoinositide 3-kinase (PI3K) family members contain one of four different catalytic subunits and generate the

    Mediating synergistic signaling

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