Cellular Signalling

The complement derived neutrophil chemoattractant C5a, is a potent activator of the neutrophil superoxide anion generating NADPH oxidase. An allosteric modulator specific for the free fatty acid 2 receptor increases the activating potency but not the efficacy of C5a. The allosteric modulator also decreases the inhibitory effect of the C5a receptor antagonist avacopan, suggesting that the NADPH oxidase is activated by two different signaling pathways downstream of the receptor for C5a. While the allosteric modulator affected the C5a-mediated activation of the NADPH oxidase, the C5a-induced rise in the intracellular concentration of free calcium ions was unaffected. The C5a receptor and the free fatty acid receptor belong to the family of G protein-coupled receptors family. Our results show that the activated C5a receptors generate signals that directly activate the NADPH oxidase and allosterically modulated free fatty acid receptors which secondarily generate signals that elicit NADPH oxidase activity. This is in line with an earlier described receptor transactivation model, by which the fatty acid receptor is activated by receptor downstream signals generated by several different neutrophil receptors to which we now add the receptor for C5a. In addition, the fatty acid receptor was higher ranked than the receptor for C5a, in the neutrophil receptor hierarchy. The dual receptor trans-regulatory effects, by which the receptor for C5a activates the fatty acid receptor and by which this receptor reduces the C5a response, represent new regulatory mechanisms of importance for the NADPH oxidase activity in neutrophils.

Positive allosteric modulators for free fatty acid receptor 2 (FFA2R/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFA2R was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFA2R from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFA2R in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFA2Rs, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFA2R activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFA2R activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFA2R activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFA2R modulator. O_LIThe allosterically modulated FFA2R is transactivated by signals generated by other GPCRs. C_LIO_LIThe PAF and ATP receptors transactivate FFA2R from the cytosolic side of the membrane. C_LIO_LIThe mechanisms that regulates activation of FFA2R from outside and inside differ. C_LI

Interferon (IFN)-{gamma} is a proinflammatory cytokine with a crucial role in intercellular communication during innate and acquired immune responses. IFN-{gamma} interacts with many cell types, among which endothelial cells. Here, we show that pharmacological and low-dose kinetically activated (SKA) IFN-{gamma} exert different effects on endothelial cells by activating different signal transduction pathways. Pharmacological concentrations of IFN-{gamma} activate JAK/STAT pathway, inducing the overexpression of the CDKN1A p21, which in turn inhibits cell growth. Conversely, low-dose SKA IFN-{gamma} does not activate the canonical JAK/STAT pathway but induces the phosphorylation of ERK. ERK activation is responsible for the induction of endothelial cell migration. Interestingly, ERK activation occurs only in the presence of kinetically activate low-dose IFN-{gamma}, underlying the importance of mechanical forces to potentiate IFN-{gamma} activity.

Type I interferon (IFN) is induced in virus infected cells, secreted and it inhibits viral replication in neighboring cells. IFN is also an important player in many non-viral diseases and in the development of normal immune cells. Although the signaling pathways for IFN induction by viral RNA or DNA have been extensively studied, its mode of induction in uninfected cells remains obscure. Here, we report that inflammatory cytokines, such as TNF- and IL-1{beta}, can induce IFN-{beta} through activation of the cytoplasmic RIG-I signaling pathway. However, RIG-I is activated not by RNA, but by PACT, the protein activator of PKR. In cell lines or primary cells expressing RIG-I and PACT, activation of the MAPK, p38, by cytokine signaling, leads to phosphorylation of PACT, which binds to primed RIG-I and activates its signaling pathway. Thus, a new mode of type I IFN induction by ubiquitous inflammatory cytokines has been revealed. Key pointsO_LICytochalasin D followed by TNF- / IL-1{beta} treatment activates IFN-{beta} expression. C_LIO_LIIFN-{beta} expression happens due to activation of RIG-I signaling. C_LIO_LIInteraction between RIG-I and PACT activates IFN-{beta} expression. C_LI

14-3-3 protein family binds and regulate hundred of serine/threonine phosphorylated proteins. Considered as redundant, ubiquitous and constantly expressed this protein family was treated as an accessory for many signaling systems. Here we studied the reversible inhibition by acetylation of its essential N-{varepsilon}-lysine 49/51 residue during the osteogenic differentiation of human adipose-derived stem cells (ASC). We found that during the differentiation of ASC the levels of 14-3-3 acK49/51 increase showing that inhibition of 14-3-3 is necessary for this process. Among the 7 paralogs of this family, the inhibition by this posttranslational modification occurs mostly on the paralog {beta} located specifically in the nucleus where 14-3-3 was described to binds to H3 histone and many transcription factors. Short hairpin RNA silencing of 14-3-3{beta} gene but not 14-3-3{gamma} increases significantly the osteogenic potential of the cells. These results show that specifically 14-3-3{beta} is a negative regulator of osteogenesis and its inhibition by acetylation on lysine 51 is the cellular mechanism to regulate it.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

ObjectiveThe uncoupling protein 1 (UCP1) is induced in brown or "beige" adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPAR{gamma} agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously. MethodsAdipocytes generated from primary human progenitor cells were differentiated without exposure to PPAR{gamma} agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses. ResultsUCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that Rsgs effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPAR{gamma} agonists. ConclusionUCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPAR{gamma}-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPAR{gamma} in response to cAMP occurs as a result of the production of natural PPAR{gamma} activating ligands through ATGL-mediated lipolysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/607465v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18675a5org.highwire.dtl.DTLVardef@95cb66org.highwire.dtl.DTLVardef@5cb189org.highwire.dtl.DTLVardef@18bac05_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myofibroblast differentiation, characterized by accumulation of cytoskeletal and extracellular matrix proteins by fibroblasts, is a key process in wound healing and pathogenesis of tissue fibrosis. Transforming growth factor-{beta} (TGF-{beta}) is the most powerful known driver of myofibroblast differentiation. TGF-{beta} signals through transmembrane receptor serine/threonine kinases that phosphorylate Smad transcription factors (Smad2/3) leading to activation of transcription of target genes. Heterotrimeric G proteins mediate a distinct signaling from seven-transmembrane G protein coupled receptors, not commonly linked to Smad activation. We asked if G protein signaling plays any role in TGF-{beta}-induced myofibroblast differentiation, using primary cultured human lung fibroblasts. Activation of Gs by cholera toxin blocked TGF-{beta}-induced myofibroblast differentiation without affecting Smad2/3 phosphorylation. Inhibition of Gi by pertussis toxin, or siRNA-mediated combined knockdown of Gq and G11 had no significant effect on TGF-{beta}-induced myofibroblast differentiation. A combined knockdown of G12 and G13 resulted in a drastic inhibition of TGF-{beta}-stimulated expression of myofibroblast marker proteins (collagen-1, fibronectin, smooth-muscle -actin), with siG12 being significantly more potent than siG13. Mechanistically, a combined knockdown of G12 and G13 resulted in a substantially reduced phosphorylation of Smad2 and Smad3 in response to TGF-{beta}, which was accompanied by a significant decrease in the expression of TGF{beta} receptors (TGFBR1, TGFBR2) and of Smad3 under siG12/13 conditions. In conclusion, our study uncovers a novel role of G12/13 proteins in the control of TGF-{beta} signaling and myofibroblast differentiation.

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Binding of uPA to uPAR localizes proteolytic cascade activation at the cell surface and can induce intracellular signaling. As uPAR possesses no transmembrane domain, it relies on uPAR cross-talk with various membrane receptors. Though uPAR role in inflammatory processes is well documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. We show that downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NF{kappa}B, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR-/- mice demonstrated strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

FGF1 is known as an anti-inflammatory and has suppresses insulin resistance. Its homologue FGF2 is pro-inflammatory. Mechanism of FGF1s anti-inflammatory action and FGF2s pro-inflammatory action are unknown. Several inflammatory cytokines (e.g., CX3CL1, CCL5, and CXCL12, and CD40L) bind to the classical ligand (RGD)-binding site (site 1) of integrin v{beta}3. In addition, they bind to the allosteric site (site 2) of v{beta}3, which is distinct from site 1, and allosterically activate v{beta}3. Site 2 is involved in inflammatory signals since inflammatory lipid mediator 5-hydroxycholesterol binds to site 2 and induces integrin activation and inflammatory signals (e.g., TNF and IL-6 secretion). We thus hypothesized that FGF1 and FGF2 bind to site 2 and affect activation status of integrins. Here we describe that FGF2 bound to site 2 and allosterically activated v{beta}3 integrin. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for inducing integrin activation. In contrast, FGF1 bound to site 2 but did not activate v{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. These findings suggest that FGF1s anti-inflammatory action is mediated by blocking site 2. FGF1 has potential as an anti-inflammatory agent, but is not appropriate for long-term use since it is potent mitogen. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed v{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for inflammatory diseases.

Macrophage polarization plays an important role in tissue regeneration. Numerous factors and signaling molecules affect polarization processes. Here we investigated the consequences of the genetic deletion of vasodilator-stimulated phosphoprotein (VASP), which increases macrophage M1 polarization through augmented signal transducer and activator of transcription 1 (STAT1) signaling, and AMP-activated protein kinase (AMPK), which attenuates inflammation by inhibiting STAT1 expression and signaling. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNF and IL-1{beta} was increased in these cells. Consistent with the role of AMPK in macrophage phagocytosis, VASP-/- macrophage phagocytosis was also significantly impaired. Interestingly, impaired phagocytosis could be rescued by exogenous activation of AMPK. Mechanistically, we found that VASP binds directly to protein phosphatase 1 regulatory subunit 6 (PP1-R6) and we hypothesize that VASP-binding to PP1- R6/PP1 limits the PP1-dependent de-phosphorylation of AMPK in wild-type cells. Conversely, AMPK dephosphorylation by the PP1-R6/PP1 complex is enhanced in the absence of VASP. In summary, we have identified a link between VASP and AMP-activated protein kinase (AMPK) activity, which may contribute to the pro-inflammatory phenotype of VASP-deficient macrophages.

Background and aimsTLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages. MethodsAdult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN{gamma}, S1/IFN{gamma} or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2. ResultsTLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid(R)), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore(R), suggesting ACE2 expression is mediated by the alternative endosomal/{beta}-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-{gamma} treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1{beta} and IL-6 mRNA. ConclusionsThese results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid(R)) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

Several vasoprotective functions of high-density lipoproteins (HDL) on the endothelium have been shown to depend on the presence of sphingosine-1-phosphate (S1P) receptors (S1PRs) as well as scavenger receptor class B type 1 (SR-B1). Interference with the presence of S1P or the activity of S1PR1 or S1PR3 mimics many effects seen by the interference with SR-B1. This raises the question on interactions between S1P receptors and SR-B1. We investigated the influence of S1PRs on SR-B1 expression in human aortic endothelial cells. Silencing or pharmacological inhibition of S1PR1 or S1PR3 down-regulated SCARB1 mRNA expression as well as SR-B1 protein abundance. RNA interference with S1PR1 or S1PR3 also decreased cellular association of 125I-HDL with HAECs. Further mechanistic studies showed that knockdown of S1PR1 or S1PR3 reduced SR-B1 protein by inducing its degradation through deceasing Akt activity. Moreover, silencing of S1PR1 or S1PR3 suppressed SCARB1 mRNA expression by decreasing cellular cAMP levels. In conclusion, we provide evidence for an as yet unappreciated interaction, namely the regulation of SR-B1 abundance by S1PRs on both transcriptional and post-translational levels, suggesting that interactions of S1PRs and SR-B1 regulate signaling functions of HDL as well as uptake of lipoproteins in endothelial cells.

Samd14 is crucial for cell signaling and survival in mouse models of acute anemia. Samd14 has an N-terminal actin capping protein (CP) and a C-terminal sterile alpha motif (SAM) to coordinate stem cell factor/Kit and erythropoietin receptor signaling pathways during terminal differentiation of red blood cell precursors. Here we present new findings that Samd14 expression is needed to maintain balanced autophagy in red blood cell precursors following acute anemia. Autophagy gene signatures and protein levels are markedly altered within the context of acute anemia when red blood cell precursors accelerate the process of erythropoiesis. Samd14 interacted via its SAM domain with phosphatidylinositol 3-phosphate (PI3P) which is an integral component of endosomal and autophagic membranes. We tested PI3Ps role in red blood cell differentiation using a small molecule inhibitor of the Class III PI 3-kinase VPS34, which is the sole kinase responsible for PI3P genesis. SAR405 treatment blocked red blood cell formation. In the absence of Samd14, higher doses of VPS34 inhibition were required to block erythroid differentiation. Given the critical roles of autophagy in normal differentiation, Samd14s stress-dependent activation and roles in autophagy suggest that this mechanism is needed to maintain progenitor levels and balance the production of healthy, mature red blood cells. Key Points- Autophagy genes/proteins are deregulated in red blood cell progenitors after hemolysis-induced acute anemia. - The anemia-activated Samd14 protein interacts with the PI3P lipid moiety which is integral to endosomal and autophagic membrane functions. - VPS34 inhibition blocks terminal erythroid maturation in a Samd14-dependent manner.

The CaV1 and CaV2 families of voltage-dependent calcium channels play a crucial role in neurotransmitter release, excitation-contraction and many other cellular processes. Comprised of the membrane pore-forming 1, intracellular {beta} and extracellular 2{delta} subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of CaV channels are attenuated by either constitutively or transiently bounds proteins in the cellular environment. The RGK (Rad, Gem, Rem, and Rem2) G-protein family potently inhibits CaV1 and CaV2 function in heterologous expression systems. RGK proteins bind to CaV{beta} and inhibit channel localization and activity by forming a ternary complex with CaV1. Here, we evaluated the influence of RGK proteins on CaV2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on CaV2.2. The G-domain and C-terminus could inhibit the CaV2.2 channel independently when co-expressed with channel subunits. Our results demonstrated that structural determinants in Gem, crucial for channel inhibition, lie within the 222-296 amino acid region containing both the partial G-domain and C-terminus as determined from chimeric CaV{beta}-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster (Dm) RGK sequences fused to CaV{beta} and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete CaV2.2 inhibition. Point mutations in the DmRGK C-terminus, conserved in mammalian RGK proteins, abrogated the CaV2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of CaV channels. Since RGK homologs are now recognized as physiological modulators in {beta}-adrenergic regulation of CaV channels, the relevance of this curious G-protein family deserves close examination.

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (M{beta}2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. The functional link between CD47 and Mac-1 was validated by co-immunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual M and {beta}2 integrin subunits, CD47 has been found to bind both subunits. Interestingly, the amount of CD47 recovered with the free {beta}2 subunit was higher than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with PMA, Mn2+, and activating antibody increased CD47 in complex with Mac-1, suggesting greater stability of the complex with integrin in the extended conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. The binding site in CD47 for Mac-1 was identified in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the {beta}2 and calf-1 and calf-2 domains of the subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

Previous studies showed that tetraspanins activate integrins, but the mechanism of this action is unclear. We previously showed that the extracellular-2 (EC2) domains of CD9, CD81, and CD151 bind to the classical RGD-binding site (site 1) of integrin v{beta}3, suggesting that they are integrin ligands. We showed that several inflammatory cytokines (e.g., CX3CL1, CXCL12, CCL5, and CD40L) bind to the allosteric site (site 2) of integrins, which is distinct from site 1, and activate integrins (allosteric activation). 25-hydroxycholesterol, a major inflammatory lipid mediator, is known to bind to site 2 and induce inflammatory signals, suggesting that site 2 plays a role in inflammatory signaling. We hypothesized that the EC2 domains activate integrins by binding to site 2. Here we describe that docking simulation predicted that CD81 EC2 binds to site 2 of v{beta}3 and more strongly to site 2 of 5{beta}1. Peptide from site 2 bound to isolated EC2 domains, suggesting that the EC2 domains bind to site 2. The EC2 domains only weakly activated v{beta}3 but more efficiently activated cell surface integrins 5{beta}1 and 4{beta}1 on the cell surface. These results are consistent with the previous findings that these tetraspanins preferentially interact with {beta}1 integrins. The integrin activation by the EC2 domains was increased at low EC2 concentrations and reduced as EC2 concentrations increased (biphasic), which is consistent with the findings that the EC2 domains bind to two sites (site 1 and 2). We propose that the EC2 binding to site 2 is a novel target for drug discovery.

1.G-protein coupled receptor 27 (GPR27) is part of the "Super Conserved Receptors Expressed in Brain" (SREB) family, alongside GPR85 and GPR173. While the endogenous ligands and functions of SREB receptors are still unknown, GPR27 has been implicated in insulin secretion and tumorigenesis. Here, we show that substituting GPR27s C-terminus domain with that of the {beta}1 adrenergic receptor ({beta}1AR) yields a chimera with {beta}1AR-like ligand selectivity and cellular functions. Interestingly, adrenergic ligands stimulation of GPR27 inhibited EGF-induced serum-responsive element (SRE) activation, independently of G-proteins and {beta}-arrestins, through dephosphorylation of c-Src and EGFR proteins. This unique response was exclusive to GPR27, as GPR85 and GPR173 showed no similar effects. These findings suggest that GPR27 is a receptor responding to adrenergic ligands to transinhibit EGFR through an atypical signaling mechanism.

We have previously identified the novel splicing variant of mouse Ppar{gamma} (Ppar{gamma}1sv) and proposed the synergistic regulation of the early stage of adipocyte differentiation by Ppar{gamma}1sv and Ppar{gamma}2. Here, we report the finding of PPAR{gamma}-binding sites within the Ppar{gamma} gene locus and its importance in adipogenesis and propose the positive feedback regulation of Ppar{gamma}1sv and Ppar{gamma}2 expression during the adipocyte differentiation of 3T3-L1 cells.

Prenylation is a universal and irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Thus, dysregulation of prenylation contributes to multiple disorders, including cancers, vascular diseases, and neurodegenerative diseases. During prenylation, prenyltransferase enzymes tether metabolically produced isoprenoid lipids to proteins via a thioether linkage. Pharmacological inhibition of the lipid synthesis pathway by statins has long been a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein {gamma} (G{gamma}) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated G{gamma} in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged G{gamma} subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to the prenylation process and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the G{gamma} polypeptide statin sensitivity, and prenylation efficacy. Our results also show that a few hydrophobic and charged residues at the Ct are crucial determinants of a proteins prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different G{gamma} subtypes, our findings explain how and why statins differentially perturb heterotrimeric G protein signaling in specific cells and tissues. Our results may provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.