G-protein signalling pathways and oestrogen: a role of balanced maintenance in osteoblasts

Oestrogen (E2) is an important regulator of bone cell function and alterations in oestrogen levels may cause abnormal bone metabolism in vivo. In this study we examined the long term effects of 17beta-oestradiol (17beta-E2) on G-proteins and the secondary signalling pathways of phospholipase C (PLC), cyclic adenosine monophosphate (cAMP), and 1,4,5-inositol triphosphate (IP3). Cells from neonatal mouse calvariae were cultured in phenol red-free RPMI 1640 medium supplemented with charcoal stripped foetal calf serum for 192 h with either oestrogen (10(-8) M), or oestrogen withdrawal after 48 h. Cultures were stimulated for the final 48 h with IL-6 (10(-10) M), or left unstimulated. Western blot analysis was undertaken on osteoblast membrane preparations obtained by 10 mM Tris-HCl, 0.1 mM EDTA pH 7.8 and centrifugation at 40,000 x g for 2 h. For cAMP study, cells were stimulated with IL-6 for either 15 min or 30 min. Intracellular cAMP was extracted from cells and measured by ELISA methodology. For the IP3 assay, cells were stimulated with IL-6 for 20 s and IP3 levels measured using radioimmunoassay. The blots revealed increased levels of Gialpha-, and Gqalpha-proteins with oestrogen withdrawal and IL-6 stimulation. This was in comparison to cells which were unstimulated, or stimulated with IL-6 with continuous 17beta-E2, or IL-6 alone. Gsalpha expression decreased with oestrogen withdrawal compared to the control. Limited amounts of Gialpha-, Gsalpha-, and Gqalpha-proteins were identified with continuous 17beta-E2. The levels of PLC isoforms PLCbeta1-2 were not affected by the differing oestrogen conditions. The cAMP production induced by IL-6 stimulation for 30 min and withdrawal of 17beta-E2 was lower and significantly different compared to the control study (P<0.05). Also IL-6 activation with continuous oestradiol increased cAMP levels and was significantly different from the control cells (P<0.01). However, 17beta-E2 had no effect on the formation of intracellular IP3, although IL-6 significantly lowered IP3 levels in all the groups compared to the control (P<0.01). These results suggest that oestrogen modulates the signal transduction pathways of G-protein molecules, and the secondary pathways of cAMP in mouse osteoblast-like cells.     

Twists and turns in the development and maintenance of the mammalian small intestine epithelium

Experimental studies during the last decade have revealed a number of signaling pathways that are critical for the development and maintenance of the intestinal epithelium and that demonstrate the molecular basis for a variety of diseases. The Notch-Delta, Wnt, Hedge Hog, TGF-beta, and other signaling pathways have been shown to form and steadily maintain the crypt-villus system, generating the proper quantities of highly-specialized cells, and ultimately defining the architectural shape of the system. Based on the characterized phenotypes and functional defects of mice resulting from various targeted knockouts, and overexpression and misexpressions of genes, a picture is emerging of the sequence of gene expression events from within the epithelium, and in the underlying mesenchyme that contribute to the regulation of cell differentiation and proliferation. This review focuses on the contributions of multiple signaling pathways to intestinal epithelial proliferation, differentiation, and structural organization, as well as the possible opportunities for cross-talk between pathways. The Notch pathway's potential ability to maintain and regulate the intestinal epithelial stem cell is discussed, in addition to its role as the primary mediator of lineage specification. Recent research that has shed light on the function of Wnt signaling and epithelial-mesenchymal cross-talk during embryonic and postnatal development is examined, along with data on the interplay of heparan sulfate proteoglycans in the signaling process.     

Neurotrophin-regulated signalling pathways

Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB and TrkC). In addition, each neurotrophin activates p75 neurotrophin receptor (p75NTR), a member of the tumour necrosis factor receptor superfamily. Through Trk receptors, neurotrophins activate Ras, phosphatidyl inositol-3 (PI3)-kinase, phospholipase C-gamma1 and signalling pathways controlled through these proteins, such as the MAP kinases. Activation of p75NTR results in activation of the nuclear factor-kappaB (NF-kappaB) and Jun kinase as well as other signalling pathways. Limiting quantities of neurotrophins during development control the number of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. The neurotrophins also regulate cell fate decisions, axon growth, dendrite growth and pruning and the expression of proteins, such as ion channels, transmitter biosynthetic enzymes and neuropeptide transmitters that are essential for normal neuronal function. Continued presence of the neurotrophins is required in the adult nervous system, where they control synaptic function and plasticity, and sustain neuronal survival, morphology and differentiation. They also have additional, subtler roles outside the nervous system. In recent years, three rare human genetic disorders, which result in deleterious effects on sensory perception, cognition and a variety of behaviours, have been shown to be attributable to mutations in brain-derived neurotrophic factor and two of the Trk receptors.     

Osteoclast signalling pathways

The osteoclast is a monocyte-derived cell with complex regulatory control due to its role, balancing calcium homeostasis with skeletal modelling and repair. Normal differentiation requires tyrosine kinase- and tumor necrosis-family receptors, normally fms and RANK. Ligands for these receptors plus unidentified serum or cell-presented factor(s) are needed for in vitro differentiation, possibly signalling via an immune-like tyrosine kinase acceptor molecule. Osteoclast development and activity are increased by cytokines signalling through GP130, such as IL-6, by TGF-beta, and by IL-1, although these cannot replace serum. Other tyrosine kinase receptors including kit and met can augment fms signalling, and TNFs other than RANKL, including TNFalpha and TRAIL, modify RANK signalling, which is also susceptible to interference by interferons. The situation is further complicated by G-protein coupled receptors including the calcitonin receptor, by integrin or calcium-mediated signals, and by estrogen receptors, which operate in bone largely via NO downstream signals. Differentiation, activity, and survival signals merge in intracellular second messengers. These include cytoplasmic kinases of several families; differentiation pathways often terminate in Erk/Jun kinases or NF-kappaB. Key regulatory intermediates include TRAF6, src, Smad3, phosphatidylinositol-3-kinase, Jak/Stat, and the cGMP-dependent protein kinase I. There are substantial uncertainties regarding how intracellular agents connect to primary signals. The frontier includes characterization of how scaffolding/adapter proteins, such as cbl, gab, grb, p130Cas, and shc, as well as itam-containing proteins and nonreceptor tyrosine kinase adapters of the src and syk families, delimit and integrate signals of multiple receptors to bring about specific outcomes.     

Twists and turns in the function of DNA damage signaling and repair proteins by post-translational modifications

When the human genome was sequenced, it was surprising to find that it contains approximately 30,000 genes and not 100,000 as most textbooks had predicted. Since then, it became clear that evolution has favored the existence of only a limited number of genes with inducible functions over multiple genes each having specific roles. Many genes products can be modified by post-translational modifications therefore fine-tuning the roles of the corresponding proteins. DNA damage signaling and repair proteins are not an exception to this rule, and they are subject to a wide range of post-translational modifications to orchestrate the DNA damage response. In this review, we will give a comprehensive view of the recent sophisticated mechanisms of DNA damage signal modifications at the nexus of double-strand break DNA damage signaling and repair.     

Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways

The vascular wilt fungus Fusarium oxysporum causes disease in a wide variety of crops. A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was previously found to be required for plant infection. To investigate the role of the heterotrimeric G-protein beta subunit Fgb1 as a putative upstream component of the Fmk1 signalling cascade, we generated F. oxysporum strains carrying either a Deltafgb1 loss-of-function allele or an fgb1(W115G) allele that mimicks the yeast STE4(W136G) mutation resulting in insensitivity to the cognate G-protein alpha subunit. Both types of mutants showed reduced virulence on tomato plants, similar to Deltafmk1 strains. However, in contrast to the latter, Deltafgb1 mutants displayed an abnormal hyphal growth phenotype with highly elongated cells, increased tip growth, a completely straight hyphal growth axis, and reduced subapical branching. Exogenous cAMP reversed part but not all of the Deltafgb1 growth phenotypes. Likewise, expression of the fgb1(W115G) allele only partly reversed growth phenotypes and failed to restore virulence on plants, whereas reintroduction of a functional fgb1 allele fully restored the wild type phenotype. Immunoblot analysis showed that levels of Fmk1 phosphorylation in fgb1 mutants were comparable to those in the wild type strain. Our results support a model in which Fgb1 controls hyphal growth, development and virulence in F. oxysporum both through cAMP-dependent and -independent pathways.     

Twists and turns of the cation-dependent mannose 6-phosphate receptor. Ligand-bound versus ligand-free receptor.

Mannose 6-phosphate receptors (MPRs) participate in the biogenesis of lysosomes in higher eukaryotes by transporting soluble acid hydrolases from the trans-Golgi network to late endosomal compartments. The receptors release their ligands into the acidic environment of the late endosome and then return to the trans-Golgi network to repeat the process. However, the mechanism that facilitates ligand binding and dissociation upon changes in pH is not known. We report the crystal structure of the extracytoplasmic domain of the homodimeric cation-dependent MPR in a ligand-free form at pH 6.5. A comparison of the ligand-bound and ligand-free structures reveals a significant change in quaternary structure as well as a reorganization of the binding pocket, with the most prominent change being the relocation of a loop (residues Glu(134)-Cys(141)). The movements involved in the bound-to-free transition of the cation-dependent MPR are reminiscent of those of the oxy-to-deoxy hemoglobin transition. These results allow us to propose a mechanism by which the receptor regulates its ligand binding upon changes in pH; the pK(a) of Glu(133) appears to be responsible for ligand release in the acidic environment of the late endosomal compartment, and the pK(a) values of the sugar phosphate and His(105) are accountable for its inability to bind ligand at the cell surface where the pH is about 7.4.     

The selective interactions and functions of regulators of G-protein signalling

There is accumulating evidence that regulators of G-protein signalling (RGS) can have roles in signal transduction that are not related to GAP activity. Furthermore, RGSs have much more selective effects in vivo than might be anticipated from their behaviour in in vitro assays. I discuss the molecular mechanisms by which these phenomena might be explained including specific interactions between the RGS and G-protein coupled receptor, G-protein and effector.     

Receptor-independent activators of heterotrimeric G-protein signaling pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.     

Wnt signalling: the case of the 'missing' G-protein

Spermatozoa represent a highly specialized cell type, with a minimalist structure designed to fulfil a single principal function: the transport of an intact single-copy haploid genome to the site of fertilization in the oviduct, and consequent zygote formation. They have lost most of their original cytoplasm, and remaining organelles are extremely modified. One result of this is that biochemical dynamics are restricted by a lack of cytoplasmic diffusion and a dramatic compartmentalization, with an increased emphasis on the physicochemical modulation of membranes. This is also reflected in a truncated apoptotic pathway, described in this issue of the Biochemical Journal in an article by Koppers et al., which leads to a so-called 'silent response' in the female tract, whereby unused sperm are removed without inflammatory consequences that might otherwise be detrimental to the new embryo. This new study shows that sperm have not simply jettisoned unwanted cellular components, but have evolved a very appropriate systems biology adapted to the specialist role they have to perform.     

Spontaneous receptor-independent heterotrimeric G-protein signalling in an RGS mutant

Tripartite G-protein-coupled receptors (GPCRs) represent one of the largest groups of signal transducers, transmitting signals from hormones, neuropeptides, odorants, food and light. Ligand-bound receptors catalyse GDP/GTP exchange on the G-protein alpha-subunit (Galpha), leading to alpha-GTP separation from the betagamma subunits and pathway activation. Activating mutations in the receptors or G proteins underlie many human diseases, including some cancers, dwarfism and premature puberty. Regulators of G-protein signalling (RGS proteins) are known to modulate the level and duration of ligand-induced signalling by accelerating the intrinsic GTPase activity of the Galpha subunit, and thus reformation of the inactive GDP-bound Galpha. Here we find that even in the absence of receptor, mutation of the RGS family member Sst2 (refs 6-9) permits spontaneous activation of the G-protein-coupled mating pathway in Saccharomyces cerevisiae at levels normally seen only in the presence of ligand. Our work demonstrates the occurrence of spontaneous tripartite G-protein signalling in vivo and identifies a requirement for RGS proteins in preventing such receptor-independent activation.     

Crosstalk among cancer signalling pathways

Identification of c-MYC as a target of the APC pathwayHe, T-C. et al. (1998)Science 281, 1509–1512Smad3 mutant mice develop metastatic colorectal cancerZhu, Y. et al. (1998)Cell 94, 703–714