Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase Cbeta3 phosphorylation by protein kinase A

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.     

G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyzed G(o) alpha-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway.     

Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2

The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.     

Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists

Basophils stimulated with IL-3 plus C5a selectively express IL-4 and IL-13 and continuously produce leukotrienes (LT) for hours. C5a combined with IL-5 or granulocyte-macrophage colony-stimulated factor was, however, much less effective in promoting cytokine expression and a late continuous phase of LTC4 production, possibly due to lower expression levels of their receptor alpha chains. Basophils also express several chemoattractant receptors, including high levels of C5a receptors, macrophage chemotactic protein (MCP) receptors (CCR2) and eotaxin receptors (CCR3), intermediate levels of CXCR1, CXCR2 and platelet-activating factor receptors, and lower levels of N-formyl-Met-Leu-Phe (fMLP) receptors. However, among the corresponding agonists, only C5a, fMLP and much more weakly MCP1, were found to induce cytokine expression and continuous LTC4 release, and only when combined with IL-3. CCR3, which is highly expressed on basophils and has been shown to mediate strong migratory but weak release responses, does not regulate cytokine expression. The weakly expressed fMLP receptor is an efficient activator of several cell functions including LTC4 formation, while CXCR2 hardly affects basophil function despite considerable expression. Thus, chemoattractant-receptors mediate different cellular responses unrelated to their expression levels.     

Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.     

Differential regulation of the C3a and C5a receptors (CD88) by IFN-gamma and PMA in U937 cells and related myeloblastic cell lines

We have analyzed the induction of the receptor for the anaphylatoxic peptide C3a (C3aR) by the immunomodulator IFN-gamma, the phorbol ester PMA, and dibutyryl cAMP (Bt2cAMP) in comparison with the C5a receptor (C5aR, CD88). For U937 cells, IFN-gamma and Bt2cAMP up-regulated the C3aR to the same extent, whereas Bt2cAMP was 20-fold more effective in C5aR induction. PMA increased the expression of the C5aR, and acted synergistically with IFN-gamma. In contrast, PMA did not increase specific 125I-hC3a binding, and actually antagonized C3aR induction by IFN-gamma. Two related human cell lines of the myeloblastic/monocytic lineage, HL-60 and Mono Mac 6, showed inducibility of the C3aR similar to U937 cells. The two receptors showed subtle differences in signal transduction. Despite comparable numbers of both receptors, IFN-gamma potentiated activation of the C5aR but not the C3aR, as measured by an increase in free cytosolic Ca2+ upon ligand activation. Interestingly, Bt2cAMP-treatment led to a functional response to C3a in U937 cells. Such differences in receptor regulation and signaling might underlie the partly differing physiologic effects of C3a and C5a on, for example, chemotaxis, induction of oxidative burst, or immunoregulatory functions.     

Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.     

Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq

The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.     

Effects of an agonist, allosteric modulator, and antagonist on guanosine-gamma-[35S]thiotriphosphate binding to liposomes with varying muscarinic receptor/Go protein stoichiometry

We investigated whether alcuronium, an allosteric modulator of muscarinic acetylcholine receptors, can induce receptor-mediated activation of Go proteins in liposomal membranes incorporating purified M2 receptors and Go proteins and whether its action is affected by the receptor/Go protein (R/Go) ratio. The binding of guanosine-gamma-[35S]thiotriphosphate ([35S]GTPgammaS) served as the indicator of G protein activation. It was stimulated by empty receptors at high receptor densities, and the dose-response curve was shifted to the left by the agonist carbachol and to the right by the antagonist atropine. At an R/Go ratio of 300:100, the rate of [35S]GTPgammaS binding was the same in the presence or absence of 0. 1 mM carbachol. Alcuronium increased the binding of [35S]GTPgammaS at R/Go ratios of <3:100 and diminished it at R/Go ratios of >10:100, similar to previous observations on intact cells expressing muscarinic receptors at different densities. The apparent biphasicity of alcuronium action indicates that the allosteric modulator has at least two effects on muscarinic receptor/G protein interaction but its mechanistic basis is unclear. The "active state" of muscarinic receptors induced by alcuronium probably is different from that induced by carbachol. Changes in the densities of receptors and Go proteins had little effect on the kinetics of [35S]GTPgammaS binding and on receptor affinity for carbachol, provided the R/Go ratio was kept constant. This suggests that the receptors and G proteins are located in microdomains in which their concentrations remain constant, despite variations in the amounts of lipidic membranes in the system.     

Neutrophils stimulated with a variety of chemoattractants exhibit rapid activation of p21-activated kinases (Paks): separate signals are required for activation and inactivation of paks

Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the alpha and betagamma subunits of complex G proteins.     

Activation of endothelin ETA receptors induces phosphorylation of alpha1b-adrenoreceptors in Rat-1 fibroblasts

The effect of endothelin-1 on the phosphorylation of alpha1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal alpha1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 approximately 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced alpha1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on alpha1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPgammaS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce alpha1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces alpha1b-adrenoreceptor phosphorylation and alters G protein coupling.     

A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor

The chemokine receptor CXCR4 is the major coreceptor used for cellular entry by T cell- tropic human immunodeficiency virus (HIV)-1 strains, whereas CCR5 is used by macrophage (M)-tropic strains. Here we show that a small-molecule inhibitor, ALX40-4C, inhibits HIV-1 envelope (Env)-mediated membrane fusion and viral entry directly at the level of coreceptor use. ALX40-4C inhibited HIV-1 use of the coreceptor CXCR4 by T- and dual-tropic HIV-1 strains, whereas use of CCR5 by M- and dual-tropic strains was not inhibited. Dual-tropic viruses capable of using both CXCR4 and CCR5 were inhibited by ALX40-4C only when cells expressed CXCR4 alone. ALX40-4C blocked stromal-derived factor (SDF)-1alpha-mediated activation of CXCR4 and binding of the monoclonal antibody 12G5 to cells expressing CXCR4. Overlap of the ALX40-4C binding site with that of 12G5 and SDF implicates direct blocking of Env interactions, rather than downregulation of receptor, as the mechanism of inhibition. Thus, ALX40-4C represents a small-molecule inhibitor of HIV-1 infection that acts directly against a chemokine receptor at the level of Env-mediated membrane fusion.     

A transmembrane domain-derived peptide inhibits D1 dopamine receptor function without affecting receptor oligomerization

In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the beta2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the beta2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384-16392). Treatment of D1DR with the D1DR transmembrane 6 peptide resulted in a dose-dependent, irreversible inhibition of D1DR antagonist binding, an effect not seen in D1DR with peptides based on transmembrane domains of other G protein-coupled receptors. Incubation with the D1DR transmembrane 6 peptide also resulted in a dose-dependent attenuation of both dopamine-induced [35S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and receptor-mediated dopamine stimulation of adenylyl cyclase activity. Notably, GTPgammaS binding and cAMP production were reduced to levels below baseline, indicating blockade of ligand-independent, intrinsic receptor activity. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists.     

Transient phosphorylation of the V1a vasopressin receptor

The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.     

Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein alpha subunits, Galphas or Galphai1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Galpha conformation. To study the possibility of tubulin involvement in Galphaq-mediated phospholipase Cbeta1 (PLCbeta1) signaling, the m1 muscarinic receptor, Galphaq, and PLCbeta1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCbeta1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCbeta1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Galphaq and PLCbeta1, accompanied by guanine nucleotide transfer from tubulin to Galphaq, is suggested as a mechanism for the enzyme activation. The PLCbeta1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCbeta1 by tubulin, since high tubulin concentrations might prevent the access of PLCbeta1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Galphaq for tubulin. Involvement of tubulin in the articulation between PLCbeta1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.     

Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4-CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.     

Response of SCC-12F, a human squamous cell carcinoma cell line, to complement attack

Serotonin 5-HT1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of G-proteins can be determined by agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT1A receptor activation of G-proteins using 8-OH-DPAT-stimulated [35S]GTPgammaS binding in membranes and brain sections. In hippocampal membranes, 8-OH-DPAT stimulated [35S]GTPgammaS binding by twofold, with an ED50 value of 25 nM. 5-HT1 antagonists, but not 5-HT2 antagonists, increased the ED50 of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [35S]GTPgammaS binding showed that 8-OH-DPAT induced the formation of high affinity [35S]GTPgammaS binding sites with a KD for GTPgammaS of 3.2 nM. [35S]GTPgammaS autoradiography, performed in brain sections with the 5-HT1A agonist 8-OH-DPAT, revealed high levels of 5-HT1A-stimulated [35S]GTPgammaS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT1A-stimulated [35S]GTPgammaS binding in sections was blocked by the addition of the 5-HT1 antagonist methiothepin. These results show that the use of agonist-stimulated [35S]GTPgammaS autoradiography for the 5-HT1A receptor system should provide new information regarding signal transduction in specific brain regions.     

Galphai is not required for chemotaxis mediated by Gi-coupled receptors

Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein betagamma subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gbetagamma, we tested the hypothesis that chemotaxis also requires activated alpha subunits (Galphai) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Galpha, Galphaqz5, which resembles Galphai in susceptibility to activation by Gi-coupled receptors but cannot regulate the Galphai effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Galphai but not that of Galphaqz5. We infer that chemotaxis does not require activation of Galphai. Because chemotaxis is mediated by Gbetagamma subunits released when Gi-coupled receptors activate Galphaqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Galphai.     

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state

Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists.     

Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells

Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.