Β1 Complex Research Articles

A 3D Structure Model of Integrin α4 β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4 β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4 β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4 β1, we began constructing a structure model for the integrin α4 β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg 2+ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4 β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/ β7 ligand binding selectivity.

8-Cysteine TGF-BP structural motifs are the site of covalent binding between mouse LTBP-3, LTBP-2, and latent TGF- β1

The small latent TGF- β complex often is associated with the latent TGF- β binding protein (LTBP). Three LTBPs (LTBP-1, -2, and -3) have been isolated to date. Previous studies have shown that LTBP-1 binds the small latent TGF- β1 complex through a disulfide bond between an 8-cysteine structural motif of LTBP-1 (TGF-bp repeat) and the propeptide dimer of latent TGF- β1 (TGF- β1 latency associated peptide). There is indirect evidence that LTBP-2 and LTBP-3 also bind the latent TGF- β complex, but the nature and location of the binding interaction are unknown. We have used immunoprecipitation, SDS-PAGE, and autoradiography to characterize the association between mouse LTBP-3 and the small latent TGF- β1 complex. We report that the second and third TGF-bp repeats of LTBP-3 covalently bind the latent complex, and we show a similar capability for the homologous TGF-bp repeats of mouse LTBP-2. The second TGF-bp repeat of LTBP-3 is unusual in that it has 9 cysteine residues instead of 8, and our results provide the first evidence that a TGF-bp repeat with an odd number of cysteine residues can covalently bind latent TGF- β1. Altogether, these results have important implications for TGF- β biosynthesis and the regulation of TGF- β activity.

Expression of beta 1 integrin complexes on the surface of unfertilized mouse oocyte.

Integrins are a family of cell surface receptors that mediate cell-cell and cell-matrix interactions in a variety of different cellular systems. Here we show that unfertilized mouse oocytes express beta 1 class integrins both at mRNA and protein levels. Using the reverse transcription polymerase chain reaction and oligonucleotide primers based on the DNA sequence of mouse integrins, the RNA transcripts for the beta 1, alpha 5 and alpha 6 subunits were detected in unfertilized oocytes. The expression of the mRNAs is paralleled by the expression of the corresponding proteins, in fact, the alpha 5/beta 1 and the alpha 6/beta 1 complexes can be immunoprecipitated with specific antibodies form 125I-surface-labeled oocytes. Using subunit-specific antibodies we also demonstrate the presence of the alpha 3/beta 1 at the oocyte surface but alpha 1, alpha 2, alpha 4 or alpha V subunits were not detectable. Since the mouse alpha 3 DNA sequence is not available, we have not tested for the corresponding transcript. Integrin subunits alpha 6 and beta 1 were differently distributed on the oocyte surface, as visualized by immunofluorescence staining and by immunoelectron microscopy. alpha 6 antigen was mainly confined to the microvillous area of the oocyte surface, while beta 1 was more homogeneously distributed over the whole oolemma. These data demonstrate for the first time the expression of three beta 1 integrin complexes in unfertilized mouse oocytes. Such proteins may have a role in sperm-egg interaction or during very early steps of embryogenesis.

Preparation and Properties of “β1 and β2”-Isomers of the Salicylato(triethylenetetramine)cobalt(III) Complex

Abstract β-Salicylato(triethylenetetramine)cobalt(III) chloride monohydrate has been obtained from salicylic acid and β-dichloro(triethylenetetramine)cobalt(III) chloride at pH 10–11. The separation of “β1 and β2” isomers was attempted by using ion exchange. The IR spectra of the “β1 and β2” complexes showed four strong absorption peaks in the 990–1100 cm−1 region. This indicates that the complexes assume the β-form. The 1H-NMR spectrum of ammine protons of coordinated triethylenetetramine of the β1 complex showed six signals with an intensity ratio of 1:1:1:1:1:1, while that of the β2 complex showed five signals with an intensity ratio of 1:1:1:1:2, in 1.8 mol dm−3 D2SO4. The order of the chemical shifts in ppm is N(2)H<N(3)H<N(4)H2<N(1)H2. Thus, the protons of the secondary N(3)H of coordinated triethylenetetramine absorb at lower fields than the terminal N(1)H2 protons. The 13C-NMR spectra of the “β1 and β2” complexes in heavy water showed six signals for trienligand carbons and seven signals for salicylato-ligand carbons. The spectra of these complexes have shown two different configurations of the “β1 and β2” complexes.