Androgen-induced growth factor and its receptor: Demonstration of the androgen-induced autocrine loop in mouse mammary carcinoma cells

SC-3 cells derived from mouse mammary carcinoma (Shinogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18–24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matris. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant from of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant from of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.     

Regulatory dendritic cells pulsed with carbonic anhydrase I protect mice from colitis induced by CD4+CD25- T cells

Inflammatory bowel disease (IBD), which is characterized by a dysregulated intestinal immune response, is postulated to be controlled by intestinal self-antigens and bacterial Ags. Fecal extracts called cecal bacterial Ag (CBA) have been implicated in the pathogenesis of IBD. In this study, we identified a major protein of CBA related to the pathogenesis of IBD and established a therapeutic approach using Ag-pulsed regulatory dendritic cells (Reg-DCs). Using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, carbonic anhydrase I (CA I) was identified as a major protein of CBA. Next, we induced colitis by transfer of CD4(+)CD25(-) T cells obtained from BALB/c mice into SCID mice. Mice were treated with CBA- or CA I-pulsed Reg-DCs (Reg-DCs(CBA) or Reg-DCs(CA1)), which expressed CD200 receptor 3 and produced high levels of IL-10. Treatment with Reg-DCs(CBA) and Reg-DCs(CA1) ameliorated colitis. This effect was shown to be Ag-specific based on no clinical response of irrelevant Ag (keyhole limpet hemocyanin)-pulsed Reg-DCs. Foxp3 mRNA expression was higher but RORγt mRNA expression was lower in the mesenteric lymph nodes (MLNs) of the Reg-DCs(CA1)-treated mice compared with those in the MLNs of control mice. In the MLNs, Reg-DCs(CA1)-treated mice had higher mRNA expression of IL-10 and TGF-β1 and lower IL-17 mRNA expression and protein production compared with those of control mice. In addition, Reg-DCs(CBA)-treated mice had higher Foxp3(+)CD4(+)CD25(+) and IL-10-producing regulatory T cell frequencies in MLNs. In conclusion, Reg-DCs(CA1) protected progression of colitis induced by CD4(+)CD25(-) T cell transfer in an Ag-specific manner by inducing the differentiation of regulatory T cells.     

A relationship between motilin and growth hormone secretagogue receptors

The motilin receptor (MR) belongs to a family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptor (GHSR). Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the MR were important for responses to the natural peptide ligand, motilin, and the transmembrane domains of the MR were important for a non-peptidyl ligand, erythromycin. We also reported that the perimembranous residues in the second extracellular loop of the GHSR were critical for natural ligand ghrelin binding and activity. The MR is 52% identical to GHSR, with 86% sequence identity in the transmembrane domains. In the current work, to gain insight into a relationship between MR and GHSR, we studied functional responses to motilin, erythromycin and ghrelin of expression cells of chimeric constructs of MR and GHSR and co-expression cells of both MR and GHSR. We also generated human MR transgenic mice, and clarified a relationship between motilin and ghrelin. MR(1-62)/GHSR(68-366) construct responded only to ghrelin, MR(1-102)/GHSR(108-366) responded to ghrelin and erythromycin, and MR(1-129)/GHSR(135-366) and MR(1-178)/GHSR(184-366) responded to erythromycin, while GHSR(1-183)/MR(179-412) responded to neither motilin, erythromycin nor ghrelin. MR and GHSR co-expression cells have no additional responses to these ligands. Motilin or erythromycin administration to human MR transgenic mice resulted in a decrease of serum acyl-ghrelin levels, while MR and GHSR mRNA expression in the gastrointestinal tracts were not changed. These data suggested that in species expressing both motilin-MR and ghrelin-GHSR, there is a compensatory relationship in vivo.     

Superchannel of bacteria: biological significance and new horizons

Sphingomonas species A1 is a newly identified pit-forming bacterium that directly incorporates a macromolecule (alginate) into its cytoplasm through a pit-dependent transport system, which we termed a superchannel. A pit is a novel, high-dimensional organ acquired through the fluidity and reconstitution of cell surface molecules, including flagellin, and through cooperation with the transport machinery in the cells, which confers upon bacterial cells a more efficient way to secure and assimilate macromolecules. The analysis of the superchannel changes general ideas regarding the fluidity and function of the cell surface, evolution and origin of cell-surface organs, including flagella, transport, and assimilation systems of macromolecules, and the divergence and energetics of metabolism.     

Mutational analysis of predicted intracellular loop domains of human motilin receptor

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63-66, 135-137, and 296-301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.     

A novel structural fold in polysaccharide lyases: Bacillus subtilis family 11 rhamnogalacturonan lyase YesW with an eight-bladed beta-propeller

Rhamnogalacturonan (RG) lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a beta-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5 A resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed beta-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a beta-propeller fold in polysaccharide lyases and provides structural insights into substrate binding by RG lyase.     

Conservation and divergence on plant seed 11S globulins based on crystal structures

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with T_m values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.     

Differential contributions of motilin receptor extracellular domains for peptide and non-peptidyl agonist binding and activity

The family of G protein-coupled receptors that includes receptors for motilin, ghrelin, and growth hormone secretagogue has substantial potential importance as drug targets. Understanding of the molecular basis of hormone binding and receptor activation should provide insights that are helpful in the development of such drugs. We previously examined the unique second extracellular loop domain of the motilin receptor, identifying key epitopes in perimembranous locations at each end of this long loop (Matsuura, B., Dong, M., and Miller, L. J. (2002) J. Biol. Chem. 277, 9834-9839). Here, we have extended that work, examining the other predicted extracellular domains of the motilin receptor by using sequential deletions of segments ranging from one to six amino acid residues and site-directed alanine replacement mutagenesis approaches. Each construct was transiently expressed in COS cells, and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and motilin radioligand binding. Only those receptor segments that included key Cys residues in positions 25, 30, and 111 or perimembranous regions at the ends of the amino terminus and the first and third extracellular loops disrupted motilin biological activity. Each of these Cys deletions also disrupted action of erythromycin. Alanine replacements for each of the potentially important amino acid residues in the perimembranous segments revealed that residues Gly36, Pro103, Leu109, and Phe332 were responsible for the selective negative impact on motilin biological activity, while responding normally to erythromycin. These results support the presence of functionally important disulfide bonds in the motilin receptor ectodomain and demonstrate that the structural determinants for binding and biological activity of peptide and non-peptidyl agonist ligands are distinct, with a broad extracellular perimembranous base contributing to normal motilin binding.