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(2518-2519) Structure and Function of Membrane Proteins II Wednesday. Degradation of Extracellular Matrix (2518-2519) 433a 2516 2519 SIMILARITY BETWEEN GROUP-I PHOSPHOLIPASE A2(PLA2) AND MANNOSE INFLUENCE HYPOXIC HUMAN RECEPTORS AND PLA2-INDUCED INVASION OF ECM BY NIH 3T3 CELLS ((A.Krtolica1 J.W.Ludlow''2)) ((G. C. Kundu and A. B. Mukherjee)), Section on Developmental Genetics, Dept. Biochemistry; HGB/NICHD/NIH, Bethesda, MD 20892. Center, Rochester, Rochester, (Spon. Hilf.) Phospholipases A2 (PLA,s) are a class of key esterases involved in the production of proinflanmatory lipid mediators. Group I induces several cellular responses, Ischemic PLA, microenvironments. Hypoxic including cell proliferation, chemokinesis and smooth muscle contraction, via receptor- microenvironments a mediated pathway. Here, we report that InI-PLA27I (porcine pancreatic) binds with responsiveness receptor on NIH 3T3 cell surface with high affinity and specificity and this binding is subpopulations displaced by cold porcine pancreatic and Nqja noja PLA2s but not by bee venom and tensions Cotalw adamas PLA2s. Scatchard analysis of competition binding of mzI-PLA27I chemotherapy, we with cold porcine pancreatic or N4ja-naja PLA2s using NIH 3T3 cells yielded stress properties We characterized nM dissociation constants (K,) of 0.5 and 2.25 nM respectively. Affinity crosslinking cells by gelatin zymography, vitro of I25-PLA2-I on NIH 3T3 cells using disuccinimidyl suberate (DSS) followed by SDS- assays. PAGE and autoradiography, identified a receptor with a molecular mass of 180 kDa. activity vitro This radiolabeled receptor band was virtually abolished when competed with cold in some but pancreatic and Naja n4ia PLA2s. Interestingly, competition affinity-crosslinkling that at populations experiments with mannose-BSA, galactose-BSA and N-acetylglucose-BSA suggested metastasis even oxygen to repopulation patient can from the receptor but there were that these ligands specifically displace m2BI-PLA27I even surgical no displacement when mannan, mannose, glucose, fucose and N-acetyl glucosamine tissue. were used. Deglycosylation (by PNGase-F) of the receptor after cross-linking drastically reduced the molecular mass (M, -150). However, deglycosylation before binding showed no radiolabeled receptor band indicating that carbohydrate moieties are required for PLA2 binding. Moreover, there was a dramatic increase in cellular invasion of artificial ECM (Matrigel") when NIH 3T3 cells were treated with PLA2-I. Our results suggest that:(i) PLA2-I receptor is present on NIH 3T3 cells and this receptor may bind ligands other than porcine pancreatic PLA5;(ii) N-glycosylation is essential for PLA2 binding and (iii) since PLA5-I is found in circulation, receptor- mediated invasion by NIH 3T3 cells may have physiological significance. Structure and Function of Membrane Proteins II (2520-2523) 2520 2521 CONTROL OF GLUT1 AND GLUT3 GLUCOSE TRANSPORTERS IN A BICARBONATE DEPENDENT H+-EXCHANGER IS UPREGULATED IN CHO CHICKEN EMBRYO FIBROBLASTS. ((P. Wagstaff, H.Y. CELLS ADAPTED TO GROWTH AT pHe 6.7. ((RA Coss, DB Leeper, ML Wahl and Kang, P.J. Robbins and M.K. White)) Department of CS Owen)) Thomas Jefferson University, Philadelphia, PA 19107. Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC Heat sensitization of mammalian cells in culture by low extracellular pH (pHe) is attributed to a reduced intracellular pH (pHi). However, it has been reported that 27858. explanted tumor cells are not sensitized to hyperthermia at reduced pHe. Also, mammalian cells adapted to growth in reduced pH medium become less sensitive to When chicken embryo fibroblasts (CEFs) are heat killing at the reduced pH. This decreased heat sensitization may result transformed by the v-src oncogene or stimulated because one or more of the celkular proton extrusion mechanisms are upregulated with mitogens, glucose transport was increased to maintain pHi at acceptable levels. Inhibition of the upregulated proton transport with an associated elevation in GLUT3 uRNA but no mechanism(s) should decrease the pHi in the phenotypically adapted cells and change in GLUT1 iRNA. This was the converse of resensitize them to hyperthermia at reduced pHe. We tested this hypothesis using the pattern of regulation in rodent cells where amiloride, an inhibitor of the Na+/H+ antiporter (the major proton exchanger in GLUT1 but not GLUT3 mRNA was modulated. We have mammalian cells), and CHO 10B cells adapted to growth in pH 6.7 medium. isolated and sequenced a chicken GLUT1 cDNA which, Adaptation was tested for by comparing the survival of adapted and nonadapted unlike its mammalian counterpart, was not induced cells heated at pH 6.7 and 7.3. Maximal adaptation of the heat response was this observed after 180 days of growth at pH 6.7 (in contrast to that observed for OvCa by v-src, serum or TPA. Nevertheless cDNA cells, which occurred within 10 days). Furthermore, adaptation was readily and mammalian GLUT1 have 95% amino acid sequence observed for 42°C but not for 45°C. Amiioride (0.5 mM) sensitized control and similarity and there is also a conserved 120 adapted cells comparably to 42°C at pH 7.3. However, the heat sensitization of the nucleotide subdomain in the 3'-UTR containing an adapted cells by amiboride at pH 6.7 was only a fraction of that observed for the ATTTA motif. Thus there is strong evolutionary control, nonadapted cells, inplying that the sodkirrproton arTtiporter was minimally conservation of GLUT1 structure but not of its upregulated in the adapted cells. Upreguiation of proton extrusion, measured by role in transport induction. In contrast, the analysing intracellular BCECF excitation spectra, was documented in cells grown at chicken GLUT3 isoform was induced by v-src and pH 6.7 for 180 days. The upreguiation was predominantly bicarbonate dependent mitogens in CEFs. Nuclear run-on studies showed (+0.025 pH unis in the absence of NaHCO3; +0.17 pH units in the presence of NaHCO3). These results suggest that pHi homeostasis in CHO10B cells adapted that induction of GLUT3 occurs at the level of exchangers Thus avian GLUT3 is like rodent to pHe 6.7 mainly occurs by upregulation of bicarbonate dependent transcription. (e.g., the bicarbonate-choride exchanger) other than or in addition to the amiloride GLUT1 in that its transcription is inducible. sensitive sodium/proton antiporter. (Supported by PO1CA56690 and T32CA09137 from NCI, NIH, DHHS). 2522 2523 FUNCTIONAL ROLE OF CYSTEINE RESIDUES IN HUMAN AEI, THE ERYTHROCYTE BAND 3 LIGAND IMMOBILIZES RECEPTOR AND ERYTHROCYTE MEMBRANE ANION EXCHANGE PROTEIN RIGIDEFIES MEMBRANE. ((S. McGee, D. Knowles, D. Anstee, N. ((J.R. Casey and R.R. Kopito)) Department of Biological Sciences, Stanford Mohandas, and J. A. Chasis)) Division of Cell and Molecular Biology, University, Stanford, California 94305-5020, U.S.A. Lawrence Berkeley Laboratory, Berkeley, CA 94720. Sulfhydryl-specific protein chemistry is a powerful tool to analyze protein structre Erythrocyte band 3, well-characterized as an anion channel protein, and function. After removing all cysteines from a protein, cysteines may be appears to also have the potential to regulate membrane mechanical reintroduced and used to probe specifically the structure at particular sites. We have properties. Utilizing ektacytometry and fluorescence recovery after constructed a cysteineless version of human AEI (AEIC-) in which all five photobleaching (FRAP), we measured the effect of band 3 ligand on cysteines of AEI (band 3) were mutated to serine. In this investigation we have membrane deformability and receptor lateral mobility. MoAb BRAC 18, characterized the role of native cysteines in the function of AEL. Wildtype and which binds to the 3rd extracellular loop of band 3, induced a dose- AEIC- were expressed by transient transfection of human embryonic kidney cells. dependent rigidification of the membrane. This rigidification was not AEIC- retains its ability to bind to the immobilized anion exchange inhibitor, 4- induced by binding of BRAC 18 Fab but was present after binding of the acetamido-4'-isothiocyano-stilbene-2,2' disulfonate (SITS), suggesting that its Fab fragment plus goat anti-rat IgG implying that a bivalent ligand was strscture is not grossly altered. The cytoplasmic domain of AEI anchors the cxucial for ligand-induced rigidification. To aacterize the effect of BRAC erythrocyte cytoskeleton to the plasma membrane, via ankyrin. Two of the mutated 18 binding on the lateral mobility of band 3, band 3 molecules were labeled cysteines in AElC- are in a region previously shown to be involved in ankyrin with eosin-5-maleimide (EMA) and the cells were incubated in varying binding and ankyrin binding is sensitive to the oxidation state of these cysteines. concentradons of unlabeled BRAC 18. EMA-labeled band 3 had a mobile The Kd for ankyrin binding was 14 nM for AEI and 22 nM for AEIC-, suggesting fraction of 47.5%. As the concentration of BRAC 18 incrased, the mobile that AEI cysteines are not essential components of the ankyrin binding site. The fraction of band 3 decreased in a dose-dependent fashion. At 20 ltg/ml formation of covalent dimers of AEI and AEIC- following treatment with the BRAC 18 the moblle fraction was < 1%. BRAC 18 was then labeled with homobifunctional crosslinker bis(sulfosuccunimidyl)suberate (BS3)indicates that FITC and its effect on band 3 lateral mobility analyzed. At saturating cysteine residues are not essential for the native oligomeric stucture. Fmally, anion antibody concenaions, the moble faction was < 10%. We
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