Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.

Integrins are a family of alpha/beta heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the alpha and beta subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of alphaIIbbeta3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the alpha and beta subunits of alphaIIbbeta3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the alpha- and beta-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.