Membrane Receptor Complex Research Articles

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.

Structural Biology by Mass Spectrometry: Mapping Protein Interaction Surfaces of Membrane Receptor Complexes with ICAT

Multi-component protein complexes underlie most high-order cellular functions. Elucidating the structures of these complexes and understanding how the interactions that bind them change under perturbation is a major goal of structural biology. To help in this effort, Underbakke et al provide a new twist on the proteomics technique of isotope coded affinity tagging (ICAT). In proteomics, ICAT allows relative quantification of proteins in comparable samples by reacting their exposed cysteine thiols with alkylating agents that contain either a heavy (13C) or light (12C) isotope and an affinity tag1; 2. One sample/condition is modified with the heavy tag, the other with the light, they are mixed, proteolytically cleaved into peptides, affinity purified and analyzed by mass spectrometry. Relative signals from the two mass tags report on the availability of the cysteine for modification in the two conditions. This availability reflects how much of a given cysteine residue (and its cognate protein) is there to be labeled, or in the present example, how reactive the cysteine residue is under the condition tested. Originally, the tag employed was a biotin moiety1, but this has been adapted to smaller, glucanime-based labels that can be enriched on a boronate matrix3; 4. Silverman and Harbury applied this method to the foot printing of protein accessibility and interaction surfaces4, in a manner that parallels the logic of hydrogen exchange mass spectrometery5. The current report was predicated by the author’s development of new ICAT reagents based on the glucamine backbone that are more water soluble, but have varying degrees of electrophilicity6. By measuring rates of alklyation (not just total amounts of product), the relative reactivity of cysteine residues that were specifically targeted to a protein surface could be better quantified. Here, the authors extend these studies to mapping interactions within the receptor kinase complexes that compose the bacterial chemotaxis transmembrane signaling system. Bacterial chemotaxis relys on polar clusters of transmembrane chemoreceptors coupled to the histidine kinase CheA through an adaptor protein CheW. There is great interest in the detailed architecture of the receptor arrays and how CheA activity is regulated by ligands7. Biochemical protection assays8; 9, genetic studies10; 11; 12; 13 and structural work14; 15 have probed functional interfaces in this ultrastable complex16, making it an ideal subject for testing new mapping techniques. The authors target the CheW coupling protein because it interacts with both CheA and the receptors. By substituting surface residues for cysteine and evaluating ICAT reactivity in the absence and presence of the other components they identify CheA and receptor binding regions of CheW that are largely consistent with the previous studies mentioned above. Unlike the proteomics application, careful work must be done to target the Cys residues appropriately and verify that substitution and subsequent modification does not alter activity; however, the affinity purification is a big advantage in avoiding background labeling in the complex mixture of native bacterial membranes. Importantly, the resolution of the technique is at the residue level, and has the potential to reveal how interactions between the components change subtly as they switch among activity states.

WRB is the receptor for TRC40/Asna1-mediated insertion of tail-anchored proteins into the ER membrane

Tail-anchored (TA) proteins are post-translationally targeted to and inserted into the endoplasmic reticulum (ER) membrane through their single C-terminal transmembrane domain. Membrane insertion of TA proteins in mammalian cells is mediated by the ATPase TRC40/Asna1 (Get3 in yeast) and a receptor in the ER membrane. We have identified tryptophan-rich basic protein (WRB), also known as congenital heart disease protein 5 (CHD5), as the ER membrane receptor for TRC40/Asna1. WRB shows sequence similarity to Get1, a subunit of the membrane receptor complex for yeast Get3. Using biochemical and cell imaging approaches, we demonstrate that WRB is an ER-resident membrane protein that interacts with TRC40/Asna1 and recruits it to the ER membrane. We identify the coiled-coil domain of WRB as the binding site for TRC40/Asna1 and show that a soluble form of the coiled-coil domain interferes with TRC40/Asna1-mediated membrane insertion of TA proteins. The identification of WRB as a component of the TRC (Get) pathway for membrane insertion of TA proteins raises new questions concerning the proposed roles of WRB (CHD5) in congenital heart disease, and heart and eye development.

Amino Acid Residues in the GerAB Protein Important in the Function and Assembly of the Alanine Spore Germination Receptor of Bacillus subtilis 168

The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

Advancement in researches of role of Hedgehog signal pathway in pathogenesis of pancreatic cancer

The Hedgehog (Hh) signaling pathway plays a key role in the embryonic development, which is formed by the Hedgehog(Hh) , membrane-receptor complex Patched (Ptch) and Smoothened (Smo), multifunctional transcription factors Glioma-associated oncogene homolog (Gli) and other signal molecules. The Hh signaling pathway controls a variety of developing processes, however, no or less expression is observed in the normal pancreas tissues.The mutational activation of the Hh signaling pathway is associated with tumorigenesis and metastasis of the pancreatic cancer. The aim of this review is to present a brief overview of the Hedgehog signaling pathway in the mechanism of the tumorigenesis of the pancreatic cancer. Key words: Hedgehog signaling pathway; Pancreatic cancer; Signal transduction

Soluble adenylyl cyclase mediates bicarbonate-dependent corneal endothelial cell protection

Cyclic AMP produced from membrane receptor complex bound adenylyl cyclases is protective in corneal endothelial cells (CEC). CEC also express soluble adenylyl cyclase (sAC), which is localized throughout the cytoplasm. When activated by HCO(3)(-), cAMP concentration ([cAMP]) increases by ∼50%. Here we ask if cAMP produced from sAC is also protective. We examined the effects of HCO(3)(-), pH, phosphodiesterase 4 inhibition by rolipram, sAC inhibition by 2HE (2-hydroxyestradiol), and sAC small interfering RNA (siRNA) knockdown on basal and staurosporine-mediated apoptosis. HCO(3)(-) (40 mM) or 50 μM rolipram raised [cAMP] to similar levels and protected endothelial cells by 50% relative to a HCO(3)(-)-free control, whereas 2HE, which decreased [cAMP] by 40%, and H89 (PKA inhibitor) doubled the apoptotic rate. sAC expression was reduced by two-thirds in the absence of HCO(3)(-) and was reduced to 15% of control by sAC siRNA. Protection by HCO(3)(-) was eliminated in siRNA-treated cells. Similarly, caspase-3 activity and cytochrome c release were reduced by HCO(3)(-) and enhanced by 2HE or siRNA. Analysis of percent annexin V+ cells as a function of [cAMP] revealed an inverse, nonlinear relation, suggesting a protective threshold [cAMP] of 10 pmol/mg protein. Relative levels of phosphorylated cAMP response element binding protein and phosphorylated Bcl-2 were decreased in CEC treated with 2HE or siRNA, suggesting that HCO(3)(-)-dependent endogenous sAC activity can mobilize antiapoptotic signal transduction. Overall, our data suggest a new role for sAC in endogenous cellular protection.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.

Cubilin Is Essential for Albumin Reabsorption in the Renal Proximal Tubule

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.

Definition and Characterization of an Inhibitor for Interleukin-31

Interleukin-31 (IL-31) is a recently described T cell-derived cytokine, mainly produced by T helper type 2 cells and related to the IL-6 cytokine family according to its structure and receptor. IL-31 is the ligand for a heterodimeric receptor composed of a gp130-like receptor (GPL) associated with the oncostatin M receptor (OSMR). A link between IL-31 and atopic dermatitis was shown by studying the phenotype of IL-31 transgenic mice and IL-31 gene haplotypes in patients suffering from dermatitis. In this study, we generated a potent IL-31 antagonist formed by external portions of OSMR and GPL fused with a linker. This fusion protein, OSMR-L-GPL, consisting of 720 amino acids, counteracted the binding of IL-31 to its membrane receptor complex and the subsequent signaling events involving the STATs and MAPK pathways. Neutralizing effects were found in IL-31-sensitive cell lines, including brain-derived cells and primary cultures of keratinocytes.

Calpain activates caspase-8 in neuron-like differentiated PC12 cells via the amyloid-β-peptide and CD95 pathways

The neurotoxic amyloid-β-peptide (Aβ) is important in the pathogenesis of Alzheimer's disease (AD). Calpain (Ca 2+-dependent protease) and caspase-8 (the initiating caspase for the extrinsic, receptor-mediated apoptosis pathway) have been implicated in AD/Aβ toxicity. We previously found that Aβ promoted degradation of calpastatin (the specific endogenous calpain inhibitor); calpastatin degradation was prevented by inhibitors of either calpain or caspase-8. The results implied a cross-talk between the two proteases and suggested that one protease was responsible for the activity of the other one. We now report on the previously unrecognized caspase-8 activation by calpain. In neuron-like differentiated PC12 cells, calpain promotes active caspase-8 formation from procaspase-8 via the Aβ and CD95 pathways, along with degradation of the procaspase-8 processing inhibitor caspase-8 (FLICE)-like inhibitory protein, short isoform (FLIP S). Inhibition of calpain (by pharmacological inhibitors and by overexpression of calpastatin) prevents the cleavage of procaspase-8 to mature, active caspase-8, and inhibits FLIP S degradation in the Aβ-treated and CD95-triggered cells. Increased cellular Ca 2+ per se results in calpain activation but does not lead to caspase-8 activation or FLIP S degradation. The results suggest that procaspase-8 and FLIP S association with cell membrane receptor complexes is required for calpain-induced caspase-8 activation. The results presented here add to the understanding of the roles of calpain, caspase-8, and CD95 pathway in AD/Aβ toxicity. Calpain-promoted activation of caspase-8 may have implications for other types of CD95-induced cell damage, and for nonapoptotic functions of caspase-8. Inhibition of calpain may be useful for modulating certain caspase-8-dependent processes.

Hijacking transferrin bound iron: protein-receptor interactions involved in iron transport in N. gonorrhoeae.

Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.

Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization

The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.

Seeing through protein complexes by high‐throughput FRET

fluorescence resonance energy transfer (alsoknown as F€orster-type resonance energy transfer, FRET) wasdescribed as a physical phenomenon in the middle of the20th century (1), it was an unrecognized tool in biology tillthe 1970s, when Stryer coined the term ‘‘spectroscopic ruler’’to describe the unique capability of FRET to be used as adistance measuring method (2). Despite these initial ad-vances, FRET was regarded as a research method forcomputer and engineering geeks until the 1990s. The intro-duction of fluorescent monoclonal antibodies, green fluores-cent protein derivatives, the development of FRET applica-tions for flow cytometry (3,4), digital imaging microscopy(5,6), and the wide-spread availability of fast computers andflexible evaluation softwares turned FRET into a fashionabletechnique.In FRET, a fluorescent donor interacts with an acceptormolecule which is separated by 2–10 nm from the donor.The interaction results in the transfer of the donor excita-tion energy to the acceptor manifested in, among others,quenching of donor fluorescence and enhancement, or sen-sitization of acceptor fluorescence (7). FRET efficiency issupposed to be dependent only on the donor–acceptor dis-tance, a property one expects from a ‘‘spectroscopic ruler.’’Although in most cases this approximation is acceptable,the validity of the underlying assumption of dynamic aver-aging has to be verified, because if not fulfilled, FRET alsocorrelates with the relative orientation of the donor and theacceptor (8).Early approaches of FRET directly measured the donor-sensitized emission of the acceptor by using special narrowband-pass filter sets to eliminate spectral overspill between thedonor, FRET, and acceptor channels (9). Even today, the litera-ture abounds with methods using FRET intensity (fluores-cence measured in the FRET channel) or uncalibrated FRETparameters. It is highly advisable to use the calibrated FRETef-ficiency instead of enigmatic and dubious parameters to pre-vent drawing false conclusions. As the development in thisarea accelerates, new methods have been described for theaccurate calculation of FRET efficiency between GFP variants(10), to account for the presence of free donors and acceptors(11) and to describe the proximity relationship of more thantwo epitopes (12,13). The fact that FRET reports the distancebetween the donor- and the acceptor-tagged epitopes, i.e., theconformation of the protein, in real-time in living cells madeit possible to solve the three-dimensional structure of mem-brane receptor complexes in intact cells (14). FRET is oftenused as a read-out parameter in assays in which the conforma-tion of a sensor is affected by a protease, ligand, or ion. Thecombination of three fluorophores in a single FRET-basedsensor makes simultaneous measurement of two parameterspossible (15).In the post-genomic era, the protein interactome is gain-ing more importance. Techniques used for the realization ofhigh-throughput mapping of protein interactions (e.g., yeasttwo-hybrid, fluorescence complementation) are now supple-mented by FRET-based sorting of cells in which certain pro-

Role of gp130‐mediated signalling pathways in the heart and its impact on potential therapeutic aspects

IL-6-type cytokines bind to plasma membrane receptor complexes containing the common signal transducing receptor chain gp130 that is ubiquitously expressed in most tissues including the heart. The two major signalling cascades activated by the gp130 receptor, SHP2/ERK and STAT pathways, have been demonstrated to play important roles in cardiac development, hypertrophy, protection and remodelling in response to physiological and pathophysiological stimuli. Experimental data, both in vivo and in vitro, imply beneficial effects of gp130 signalling on cardiomyocytes in terms of growth and survival. In contrast, it has been reported that elevated serum levels of IL-6 cytokines and gp130 proteins are strong prognostic markers for morbidity and mortality in patients with heart failure or after myocardial infarction. Moreover, it has been shown that the local gp130 receptor system is altered in failing human hearts. In the present review, we summarize the basic principles of gp130 signalling, which requires simultaneous activation of STAT and ERK pathways under the tight control of positive and negative intracellular signalling modulators to provide a balanced biological outcome. Furthermore, we highlight the key role of the gp130 receptor and its major downstream effectors in the heart in terms of development and regeneration and in response to various physiological and pathophysiological stress situations. Finally, we comment on tissue-specific diversity and challenges in targeted pharmacological interference with components of the gp130 receptor system.

Interleukin-6 (IL-6) and/or Soluble IL-6 Receptor Down-regulation of Human Type II Collagen Gene Expression in Articular Chondrocytes Requires a Decrease of Sp1·Sp3 Ratio and of the Binding Activity of Both Factors to the COL2A1 Promoter

Type II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-35 sequence that binds Sp1.Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6- and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription.

Wnt signaling control of bone cell apoptosis

Wnts are a large family of growth factors that mediate essential biological processes like embryogenesis, morphogenesis and organogenesis. These proteins also play a role in oncogenesis, and they regulate apoptosis in many tissues. Wnts bind to a membrane receptor complex comprised of a frizzled (FZD) G-protein-coupled receptor and a low-density lipoprotein (LDL) receptor-related protein (LRP). The formation of this ligand-receptor complex initiates a number of signaling cascades that include the canonical/beta-catenin pathway as well as several noncanonical pathways. In recent years, canonical Wnt signaling has been reported to play a significant role in the control of bone formation. Clinical studies have found that mutations in LRP-5 are associated with reduced bone mineral density (BMD) and fractures. Investigations of knockout and transgenic mouse models of Wnt pathway components have shown that canonical Wnt signaling modulates most aspects of osteoblast physiology including proliferation, differentiation, function and apoptosis. Transgenic mice expressing a gain of function mutant of LRP-5 in bone, or mice lacking the Wnt antagonist secreted frizzled-related protein-1, exhibit elevated BMD and suppressed osteoblast apoptosis. In addition, preclinical studies with pharmacologic compounds such as those that inhibit glycogen synthase kinase-3beta support the importance of the canonical Wnt pathway in modulation of bone formation and osteoblast apoptosis.

Progesterone Receptor Membrane Component-1 (PGRMC1) Is the Mediator of Progesterone’s Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells As Revealed by PGRMC1 Small Interfering Ribonucleic Acid Treatment and Functional Analysis of PGRMC1 Mutations

Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1's role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [(3)H]P4 binding and the loss of P4's antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [(3)H]P4 specifically binds to PGRMC1 at a single site with an apparent K(d) of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [(3)H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70-130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4's antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1's capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4's antiapoptotic action.

The role of the Wnt signaling pathway in osteoblast commitment and differentiation

Wnts are a large family of proteins that participate in an array of cellular biological processes such as embryogenesis, organogenesis and tumor formation. These proteins bind to membrane receptor complexes comprised of a frizzled (Fz) G-protein-coupled receptor and other membrane co-receptors forming molecular groups that initiate, at least, three different intracellular signaling cascades leading to nuclear generation of transcription factors which regulate various cellular events. These events result in selective cellular differentiation, reduction or inhibition of the apoptotic mechanisms or changes in the biologic behavior of various cell lines. During the last decade, canonical Wnt signaling has been shown to play a significant role in the control of osteoblastogenesis and bone formation. In several clinical cases, mutations have been found in the Wnt receptor complexes that are associated with changes in bone mineral density and fractures. Loss-of-function mutations in LRP5 receptors cause, osteoporosis-pseudoglioma syndrome, while gain-of-function mutations in the same group lead to high bone mass phenotypes. Furthermore, osteocytes secrete proteins such as sclerostin, which blocks the membrane complex activation by Wnt, resulting in inhibition of bone formation. Studies of knockout and transgenic mouse models for Wnt pathway components have demonstrated that canonical signaling regulates most aspects of osteoblast physiology including commitment, differentiation, bone matrix formation/mineralization and apoptosis as well as coupling to osteoclastogenesis and bone resorption. Future studies in this rapidly growing area of research can possibly lead to the identification of targets of pharmacological intervention useful in the management of osteoporosis. In the present review we summarized the current knowledge related to the various components of the Wnt signaling pathway, the ways they cooperate in inducing and directing transcriptional functions as well as the interacting points with the TGFbeta superfamily. We also outlined a probable working integrating model of the mechanism of bone formation.

Macrophage migration inhibitory factor does not modulate co-activation of androgen receptor by Jab1/CSN5

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory immune modulator that plays an important role in the regulation of innate and adaptive immune responses. MIF signaling involves CD74/CD44 membrane receptor complexes, the chemokine receptors CXCR2 and 4 as well as uptake by non-receptor mediated endocytosis. Endocytosed or endogenous MIF interacts with Jun activation domain-binding protein 1 (Jab1), originally described as transcriptional co-activator for the transcription factor AP-1, that is also known as subunit 5 of the COP9 signalosome (CSN5). Since Jab1/CSN5 also functions as a co-activator for a number of steroid hormone receptors (SHRs), it had been speculated that MIF could modulate Jab1/CSN5-SHR interactions. Here we show (i) that fluorescently labeled MIF is internalized by NIH 3T3 cells within minutes, (ii) compromises the induction of phospho-c-Jun levels by TNFalpha and PMA and, hence, is biologically active, but (iii) is not able to interfere with co-activation by Jab1/CSN5 of the androgen receptor.

Clustering of membrane proteins in the pre‐stimulation stage is required for signal transduction: a computer analysis

AbstractThe physiological significance of membrane protein clustering for signal transduction was examined theoretically using a Monte Carlo computer simulation. Simulation results revealed that pre‐stimulation clustering of membrane proteins enhanced signal transduction. Membrane protein clustering induced by the binding of external stimuli provided no kinetic advantage in terms of formation rate or maximum quantity of active membrane receptor complexes. These data suggested that membrane proteins associate weakly in the clustering areas of non‐stimulated cells, and that their association is strengthened upon binding of extracellular stimuli to the membrane receptor. Additionally, the number of cytosolic proteins recruited to membrane receptor complexes was not increased by the membrane complex clustering, except when cytosolic signal proteins were localized to a narrow area such as a tunnel that ran from the membrane cluster to the nucleus. Simulations were carried out on a conventional personal computer under Windows XP or 2000 operating systems. Since neither special computing hardware nor special training is required, our simulation procedure could be easily adapted for kinetic analysis of any signal transduction pathway.