Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Allosteric effects of chloride ions at the intradimeric alpha1beta1 and alpha2beta2 interfaces of human hemoglobin

The structural transition induced by ligand binding in human hemoglobin encompasses quaternary structure changes at the interfaces between the two alphabeta dimers. In contrast, the interfaces between alpha and beta subunits within the same dimer (i.e., alpha1beta1 and alpha2beta2 interfaces) are structurally invariant. Previous work from this laboratory using NMR spectroscopy has identified four sites at the intradimeric alpha1beta1 and alpha2beta2 interfaces that, although structurally invariant, experience significant changes in the rates of proton exchange upon ligand binding. These sites are Hisalpha103(G10) and Hisalpha122(H5) in each alpha subunit of the hemoglobin tetramer. In the present work, we show that the proton exchange at the Hisalpha103(G10) sites is affected by the interactions of hemoglobin with chloride ions. Increasing concentrations of chloride ions at pH 6.45 and at 37 degrees C enhance the exchange rate of the Hisalpha103(G10) N(epsilon 2) proton. The enhancement is greater in deoxygenated than in ligated hemoglobin. In the framework of the local unfolding model for proton exchange, these results suggest that the structural free energy and/or the proton transfer reactions at the Hisalpha103(G10) sites depend on the concentration of chloride ions. Therefore, the ligand-induced changes at the Hisalpha103(G10) sites are modulated by the allosteric effect of chloride ions on hemoglobin.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

Hemoglobin Tarrant: alpha126(H9) Asp leads to Asn. A new hemoglobin variant in the alpha1beta1 contact region showing high oxygen affinity and reduced cooperativity.

Hemoglobin (Hb) Tarrant was detected by its electrophoretic mobility on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). On cellulose acetate it moved as a band between hemoglobins F and S, and on citrate agar as a band at hemoglobin S. The test for solubility in 2 M phosphate buffer with Na2S2O4 was negative. The new variant has a substitution of asparagine for aspartic acid in position 126 of the alpha-chain, one of the sites involved in the alpha1beta1 contact. Furthermore, in deoxyhemoglobin aspartic acid 126 of each alpha chain also forms a non-covalent electrostatic salt bridge with arginine 141 of the corresponding alpha chain (Perutz, M. F. and Ten Eyck, L. F. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 295-310 and Perutz, M. F. (1970) Nature 228, 726-739). As a consequence of this substitution in hemoglobin Tarrant, the deoxy conformation or T state is destabilized because these two bridges cannot be formed. This condition is reflected in high oxygen affinity and low cooperativity.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Hemoglobin Athens-Georgia, or alpha 2 beta 2 40(C6)Arg replaced by Lys, a hemoglobin variant with an increased oxygen affinity.

A new hemoglobin variant, termed hemoglobin Athens-Georgia, has been found in a 23-year-old Caucasian student and three members of her family. The electrophoretic mobility of this variant at pH 9.0 is slightly less than that of hemoglobin-A. Arginyl residue in position 40 of the beta chain, corresponding to position 6 of the C helix, has been replaced by a lysyl residue. This amino acid substitution is at the alpha1-beta2 contact and slightly affects the oxygen binding properties of the hemoglobin molecule. Hemoglobin Athens-Georgia has an increased affinity for oxygen, a normal heme-heme interaction and a normal Bohr effect. Hematological abnormalities are not associated with this variant.     

Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state

When methionine beta(55)D6 in human hemoglobin is oxidized to its sulfoxide derivative, the modified protein appears to maintain most of the chemical and structural properties typical of the native protein. On the contrary, the functional behavior is drastically changed, being characterized (like that of the isolated chains) by high oxygen affinity (p50 = 0.47 torr in 0.1 M Tris (pH 7.3) + 0.1 M NaCl at 25 degrees C), absence of cooperativity (n = 1), and lack of Bohr effect. The complete destabilization of the T-state as a result of this modification is related to a perturbation of the alpha 1 beta 1 subunit interface, which in native hemoglobin remains static during the quaternary ligand-linked transition. Results also suggest that methionyl sulfoxide-containing hemoglobin, obtained under different conditions, assumes functionally different R-states, none of which is exactly comparable with that typical of the native protein.     

Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement

The major isoform of the gamma-aminobutyric acid type A (GABA(A)) receptor is thought to be composed of 2alpha(1), 2beta(2), and 1gamma(2) subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA(A) receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor gamma(2)beta(2)alpha(1)beta(2)alpha(1), could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type alpha(1)beta(2)gamma(2) GABA(A) receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269-276, we provide evidence for an arrangement gamma(2)beta(2)alpha(1)beta(2)alpha(1), counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT(3) receptors.     

Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1

Polymerization of the ECM proteins fibronectin and laminin has been shown to take place in close vicinity to the cell surface and be facilitated by beta(1) integrins (Lohikangas, L., Gullberg, D., and Johansson, S. (2001) Exp. Cell Res. 265, 135-144 and Wennerberg, K., Lohikangas, L., Gullberg, D., Pfaff, M., Johansson, S., and Fassler, R. (1996) J. Cell Biol. 132, 227-238). We have studied the role of collagen receptors, integrins alpha(11)beta(1) and alpha(2)beta(1), and fibronectin in collagen polymerization using fibronectin-deficient mouse embryonic fibroblast cell lines. In contrast to the earlier belief that collagen polymerization occurs via self-assembly of collagen molecules we show that a preformed fibronectin matrix is essential for collagen network formation and that collagen-binding integrins strongly enhance this process. Thus, collagen deposition is regulated by the cells, both indirectly through integrin alpha(5)beta(1)-dependent polymerization of fibronectin and directly through collagen-binding integrins.     

Allosteric free energy changes at the alpha 1 beta 2 interface of human hemoglobin probed by proton exchange of Trp beta 37

The energetic changes that occur on ligand binding in human hemoglobin have been investigated by measurements of the exchange rates of the indole proton of Trpbeta37(C3). The Trpbeta37 residues are located in helices C of the beta-subunits and are involved in contacts with the segments FG of the alpha-subunits at the interdimeric alpha1beta2 and alpha2beta1 interfaces of the hemoglobin tetramer. In the quaternary structure change that accompanies ligand binding to hemoglobin, these contacts undergo minimal changes in relative orientation and in packing, thereby acting as hinges, or flexible joints. The exchange rates of the indole proton of Trpbeta37(C3) were measured by nuclear magnetic resonance spectroscopy, in both deoxygenated and ligated hemoglobin. The results indicate that, at 15 degrees C, the exchange rate is increased from 9.0. 10(-6) to 3.3. 10(-4) s(-1) upon ligand binding to hemoglobin. This change suggests that the structural units at the hinge regions of the alpha1beta2/alpha2beta1 interfaces containing Trpbeta37(C3) are specifically stabilized in unligated hemoglobin, and experience a change in structural free energy of approximately 4 kcal/(mol tetramer) upon ligand binding. Therefore, the hinge regions of the alpha1beta2/alpha2beta1 interfaces could play a role in the transmission of free energy through the hemoglobin molecule during its allosteric transition.     

Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity- purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.     

The dissociation of carbon monoxide from the alpha and the beta subunits of human carbonmonoxy hemoglobin

We have measured the intrinsic CO dissociation rates from the subunits of the human hemoglobin tetramers (alpha CO beta NO)2 and (alpha NO beta CO)2 using microperoxidase and a stopped-flow spectrophotometer. The dissociation of NO is negligible. The rate constants for the and the subunits are similar (0.014 s-1 vs. 0.011 s-1, respectively, at pH 7, 20 C; and 0.016 s-1 for both in the presence of inositol hexaphosphate), indicating that they are equivalent in the first step of the CO dissociation. Therefore, the chain unequality observed in the third and fourth steps (Samaja, M., Rovida, E., Niggeler, M., Perrella, M., and Rossi-Bernardi, L. (1987). J. Biol. Chem.: 262, 4528-4533) are not due to the intrinsic properties of the subunits, but to the conformational state of the molecule.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

The amino acid sequences of the alpha and beta chains of hemoglobin from the snake, Liophis miliaris

The hemoglobin of Liophis miliaris has unusual properties. The hemoglobin is dimeric in the oxy form, and the cooperativity of O2 binding is very low, but both the Bohr effect and cooperativity are greatly enhanced in the presence of ATP (Matsuura, M. S. A., Ogo, S. H., and Focesi, A., Jr. (1987) Comp. Biochem. Physiol. 86A, 683-687). Four unique chains (2 alpha, 2 beta) can be isolated from the hemolysate. The amino acid sequences of one alpha and one beta chain have been determined in an effort to understand the functional properties. Comparison of the sequences with those of the alpha and beta chains of human Hb shows the following. (i) All 7 of the residues in the beta chain normally conserved in globins are identical to those of the human chain: Gly(B6), Phe(CD1), His(E7), Leu(F4), His(F8), Lys(H10), and Tyr(HC2), except that the distal His(E7) has been replaced by Gln in the alpha chain. (ii) All heme contact residues in the beta chain are identical with those in the human chain, but two differences are present in the alpha chain: the distal His(E7) is replaced by Gln and Met(B13) by Leu. (iii) All residues that form the binding site for organic phosphates are identical to those in human Hb. (iv) The major residues that contribute to the normal Bohr effect in human Hb, Asp-beta 94, His-beta 146, and Val-alpha 1 are conserved. (v) All beta chain residues at the alpha 1 beta 2 interface are identical with those in the human chain except two: Glu(G3)----Val and Glu(CD2)----Thr; these differences in charged residues may explain the dissociation to dimers. (vi) The 23 residues of the alpha chain in the alpha 1 beta 2 contact region are identical with those of the human chain except three: Phe(B14)----Leu, Thr(C3)----Gln and Pro(CD2)----Ser. (vii) A total of 17 differences occur at the alpha 1 beta 1 interface, 11 in the alpha chain and 6 in the beta chain.     

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.     

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and G? 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.