Specific leukotriene receptors couple to distinct G proteins to effect stimulation of alveolar macrophage host defense functions

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6β4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6β4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin–integrin-dependent pathway that negatively regulates myelination.     

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.     

Two different T cell receptors use different thermodynamic strategies to recognize the same peptide/MHC ligand

A6 and B7 are two alphabeta T cell receptors (TCRs) that recognize the Tax peptide presented by the class I major histocompatibility molecule HLA-A2 (Tax/HLA-A2). Despite the fact that the two TCRs have different CDR loops and use different amino acid residues to contact their ligand, both receptors bind ligand with similar diagonal orientations. Here we show that they also bind with very similar binding affinities and kinetics (the DeltaDeltaG degrees for binding is approximately 0.3kcal/mol at 25 degrees C). The two receptors respond similarly to alterations in the MHC molecule, yet differ dramatically in their responses to ionic strength and temperature. The different responses to temperature indicate markedly different binding thermodynamics, which are not predictable from the surface area buried in the interfaces. A6 and B7 thus represent two TCRs that are both compatible with Tax/HLA-A2, although compatibility has been achieved through the use of different thermodynamic strategies. Finally, neither A6 nor B7 are predicted to undergo large conformational adaptations upon binding, distinguishing them from a number of other TCRs whose structure, thermodynamics, and kinetics have been characterized.     

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.     

Comparative analysis of proteins selectively released from cell nuclei of different eukaryotic species under conditions of mild hydrolysis by nucleases

Nuclei of cultured Chinese hamster fibroblasts, dechoryonized eggs ofDrosophila melanogaster, calf thymocytes and mouse spleen were digested with micrococcal nuclease or DNAse I under conditions when about 15–20% of nuclear DNA is hydrolyzed. Proteins released from the nuclei with this DNA were analyzed by electrophoresis in polyacrylamide gels in the presence of SDS and the most prominent components were purified in each case. It is shown that these components derived from different species have different electrophoretic mobilities but are very similar in the amino acid composition characterized by high glycine content and a low lysine/arginine ratio. Molecular weights of two of those components, from mouse spleen and thymocytes, were estimated and are shown to be 25,500 and 23,000, respectively. Notwithstanding their different molecular weights they have the same N-terminal amino acid-arginine.     

Functionally different GPI proteins are organized in different domains on the neuronal surface

We have investigated the organization, on the plasma membrane and in detergent-insoluble membrane vesicles, of two neuronal glycosylphosphatidylinositol-anchored (GPI) proteins: Thy-1, a negative regulator of transmembrane signalling; and prion protein, whose rapid endocytosis and Cu(2+) binding suggest that it functions in metal ion uptake. Prion protein occurred on the neuronal surface at high density in domains, located primarily at the cell body, which were relatively soluble in detergent. Thy-1, although much more abundantly expressed on neurons, occurred at lower density over much of the surface of neurites (and in lower abundance at the cell body) in domains that were highly resistant to detergent solubilization. Detergent-insoluble membrane vesicles contained Thy-1 at a density similar to that on the neuronal surface. Vesicles containing each protein could be separated by immunoaffinity isolation; lectin binding showed that they were enriched in different glycoproteins. Our results demonstrate a structural diversity of the domains occupied by functionally different GPI proteins.     

B cell receptors and complement receptors target the antigen to distinct intracellular compartments

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.     

Functionally distinct tendons have different biomechanical,biochemical and histological responses to in vitro unloading

Tendons with different in vivo functions are known to have different baseline biomechanics, biochemistry and ultrastructure, and these can be affected by changes in loading. However it is not know whether different tendon types respond in the same, or different ways, to changes in loading.This study performed in vitro un-loading (stress deprivation) in culture on ovine medial extensor tendons (MET, a positional tendon), and superficial and deep digital flexor tendons (SDFTs and DDFTs, with energy-storing and intermediate functions respectively), for 21 days (n = 14 each). Tensile strength and elastic modulus were then measured, followed by biochemical assays for sulphated glycosaminoglycan (sGAG) and hydroxyproline content. Histological inspection for cell morphology, cell density and collagen alignment was also performed.The positional tendon (MET) had a significant reduction (∼50%) in modulus and strength (P < 0.001) after in vitro stress-deprivation, however there were no significant effects on the energy-storing tendons (SDFT and DDFT). In contrast, sGAG was not affected in the MET, but was reduced in the SDFT and DDFT (P < 0.001). All tendons lost compactness and collagen organisation, and had reduced cell density, but these were more rapid in the MET than the SDFT and DDFT.These results suggest that different tendon types respond to identical stimuli in different ways, thus; (i) the results from an experiment in one tendon type may not be as applicable to other tendon types as previously thought, (ii) positional tendons may be particularly vulnerable to clinical stress-deprivation, and (iii) graft tendon source may affect the biological response to loading in ligament and tendon reconstruction.     

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.     

Mast cell membrane antigens and Fc receptors in anaphylaxis: II. Functionally distinct receptors for IgG and for IgE on mouse mast cells

The present work was aimed at analyzing the functional relationships between mouse mast cell receptors for IgG and IgE antibodies. It was based on a study of the inhibition of IgG1-and IgE-induced passive mast cell degranulation produced by various immunoglobulin preparations capable of interfering with Fc receptors. Rat myeloma IgE, a high-affinity ligand for IgE receptors, was used to search for a possible participation of IgE receptors in IgG1-dependent degranulation. Mouse myeloma IgG, which inhibited only weakly IgG1-mediated reactions, had no chance to compete successfully with high-affinity IgE antibodies, but aggregated HGG was found to behave as a high-affinity ligand for IgG receptors. This enabled us to search for a possible participation of IgG receptors in IgE-dependent degranulation. The results show that rat myeloma IgE and aggregated HGG specifically inhibited IgE-induced and IgG1-induced reactions, respectively, but failed to inhibit reactions not requiring free Fc receptors. The conclusion was that receptors for IgG and for IgE are functionally independent on mouse mast cells, and are both expressed on the same cells.     

Accessory proteins in the biogenesis of G protein-coupled receptors

Intracellular accessory proteins can be critical for G protein-coupled receptor (GPCR) biogenesis, including aspects of GPCR trafficking. Recent discoveries include the identification of multiple membrane-associated proteins that dictate not only the intracellular sequestration and/or transport of GPCRs, but also modulate-quite dramatically-GPCR ligand specificity subsequent to delivery to the cell surface. These exciting discoveries have shifted earlier paradigms of GPCR functionality.     

The coupling of 5-oxo-eicosanoid receptors to heterotrimeric G proteins

5-Oxo-eicosatetraenoic acid (5-oxoETE) stimulated human neutrophil (PMN) and eosinophil chemotaxis, PMN hexose uptake, and PMN membrane GTP/GDP exchange. Pertussis toxin (PT), a blocker of heterotrimeric G proteins (GP), completely inhibited these responses, but proved far less effective on the same responses when elicited by leukotriene B4, C5a, FMLP, platelet-activating factor, IL-8, or RANTES chemotactic factors. 5-OxoETE also specifically bound to the membrane preparations that conducted GTP/GDP exchange. This binding was down-regulated by GTPgammaS, but not ADPgammaS, and displaced by 5-oxoETE analogues, but not by leukotriene B4, lipoxin A4, or lipoxin B4. Finally, PMN expressed PT-sensitive GP alphaiota2 and PT-resistant GP alphaq/11- and alpha13-chains; eosinophils expressed only alphai2 and alphaq/11. We conclude that 5-oxoETE activates granulocytes through a unique receptor that couples preferentially to PT-sensitive GP. The strict dependency of this putative receptor on PT-sensitive GP may underlie the limited actions of 5-oxoETE, compared with other CF, and help clarify the complex relations between receptors, GP, cell signals, and cell responses.