Activation of signalling by the activin receptor complex

Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals.     

Trypsin activation pathway of rotavirus infectivity

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.     

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.     

Knockout and reconstitution of a functional human type I interferon receptor complex

The functional subunits of the human Type I interferon (IFN) receptor complex have not been defined. Using site-specific recombination in a yeast artificial chromosome (YAC), we have produced a deletion within the human IFN-alpha receptor (Hu-IFN-alpha R1) gene which eliminates exon II of the gene. This deletion effectively eliminates the MHC Class I antigen induction and antiviral activity previously reported for this fully functional parental YAC clone (Soh, J., Mariano, T. M., Lim, J.-K., Izotova, L., Mirochnitchenko, O., Schwartz, B., Langer, J., and Pestka, S. (1994c) J. Biol. Chem. 269, 18102-18110). We have successfully reconstituted this activity by expression of the cDNA encoding the Hu-IFN-alpha R1 component (Uzé, G., Lutfalla, G., and Gresser, I. (1990) Cell 60, 225-234) in cells containing the YAC with this deletion. The Hu-IFN-alpha R1 subunit thus plays a critical role in the functional human Type I IFN receptor complex, whose components are encoded on this YAC. In addition, as binding of ligands is retained in the cells containing the YAC with the deletion, it is clear a second subunit encoded on the YAC is responsible for ligand binding activity. This system will now allow the identification of additional subunits involved in the response to the Type I IFNs and the functional significance of each.     

An improved assay for nuclear estrogen receptor in experimental and human breast cancer

We have validated a hydroxylapatite assay for measuring estrogen receptor in extracts from breast tumor nuclei. By adsorption of receptor of hydroxylapatite prior to addition of radioactive ligand and warming, receptor degradation can be avoided. Total binding sites are measured at 30 degrees by exchange, and receptor sites unoccupied by steroid are measured at 4 degrees. A single saturating dose of 5 nM tritiated estradiol (with or without a 100-fold excess of nonradioactive diethylstilbestrol to estimate nonspecific binding) yields results similar to a six-dose Scatchard plot. Following in vivo injection of estradiol into rats bearing mammary tumors, receptor translocation in the tumors can be accurately quantitated with this assay. Applying the assay to human breast cancer, we find that tumor biopsies may contain cytoplasmic receptor alone or may also have appreciable nuclear receptor. The latter may be bound to estradiol or may be found in "free" form. The finding of free receptor in the nuclei in certain cases raises the possibility that unoccupied receptor might be able to stimulate cell replication in these cases, even in the absence of estrogen.     

The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation

Immunoreceptors such as the high affinity IgE receptor, FcepsilonRI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated FcepsilonRI ITAM-containing subunits beta and gamma. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the FcepsilonRI beta or gamma ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor zeta chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5'-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. FcepsilonRI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.     

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.     

Plasma apolipoprotein VLDL-II and egg production in laying hens: establishment of an ELISA method

The thrombin receptor on platelets is an integral membrane protein and is cleaved by thrombin to expose a "tethered ligand" that binds to and triggers the receptor. Here we have explored the power of phage selection technology to make a peptide antagonist of this receptor using platelets directly for the selection. To focus the selection to the thrombin receptor, we eluted the phage with a peptide agonist of the thrombin receptor. A repertoire (1 x 10(7) phage clones) displaying peptide sequences based on the sequence of the tethered ligand, was constructed and selected by binding to the platelets. After several rounds of selection, we identified phage clones that were able to immunoprecipitate the thrombin receptor from platelets and the encoded peptides were sequenced. This revealed some features in common with the tethered ligand, in particular an arginine residue followed by a proline. Several of the peptides were synthesized chemically and one of the peptides was shown to antagonise platelet aggregation triggered by the agonist peptide, and to inhibit serotonin release and tyrosine phosphorylation triggered by either thrombin or the agonist peptide. Anti-aggregatory activity was about ten-fold higher than that of previously reported peptide antagonists of the thrombin receptor.     

Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. In this report we show that efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. Also, we demonstrate that the allosteric inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differ between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. Finally, the AF2 activation domain of both subunits contribute to activation even if only one of the receptors is associated with ligand. Our data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner.     

Synthesis, biological activity, and molecular modeling of selective 5-HT(2C/2B) receptor antagonists

The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.     

Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4-9-fold increase in [3H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP was concentration-dependent, with an EC50 of 20-35 nM in both cell lines. [D-Phe6,beta-Ala11,Phe13,Nle14]Bn-(6-14) stimulated a 2-3-fold increase in [Ca2+]i, a 3-fold increase in tyrosine phosphorylation of p125(FAK) with an EC50 of 0.2-0.7 nM, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [D-Arg1,D-Trp7,9, Leu11]substance P and [D-Pro4,D-Trp7,9,10]substance P-(4-11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a competitive fashion (Ki = 0.5 microM). Stimulation of p125(FAK) tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+]i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125(FAK), and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [D-Phe6,beta-Ala11, Phe13,Nle14]Bn-(6-14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 microM, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.     

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.     

Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

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.     

Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C

The receptor tyrosine kinase Kit and Kit ligand (KL), encoded at the murine white spotting (W) and steel (Sl) loci, respectively, function in hematopoiesis, melanogenesis, and gametogenesis. To understand the mechanism of turnover of Kit in mast cells, mutant receptors generated in vitro were heterologously expressed in Wsb/Wsh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induced internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL.Kit receptor complexes were rapidly internalized, and the turnover was accelerated by ubiquitin-mediated degradation. Inactivation of the Kit kinase resulted in a reduced rate of internalization of KL.Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of KL-induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradation of Kit. Furthermore, Kit receptors can be down-regulated by proteolytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of KL.Kit complexes and ubiquitination/degradation require an active kinase. By contrast, proteolytic cleavage of Kit mediated by protein kinase C activation is independent of kinase activity.