Interactions between transmembrane (TM) helices play an important role in the regulation of diverse biological functions. For example, the TM helices of integrins are believed to interact heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion. However, it has been difficult to demonstrate the specificity and affinity of the interaction between integrin TM helices and to relate them to the activation process. To examine integrin TM helix associations, we developed a bacterial reporter system and used it to define the sequence motif required for helix-helix interactions in the β 1 and β 3 integrin subfamilies. The helices interact in a novel three-dimensional motif, the "reciprocating large-small motif" that is also observed in the crystal structures of unrelated proteins. Modest but specific stabilization of helix associations is realized via packing of complementary small and large groups on neighboring helices. Mutations destabilizing this motif activate native, fulllength integrins. Thus, this highly conserved dissociable motif plays a vital and widespread role as an on-off switch that can integrate with other control elements during integrin activation.interaction motif | transmembrane domain

The translation release factors (RFs) RF1 and RF2 of Escherichia coli are methylated at the N 5 -glutamine of the GGQ motif by PrmC methyltransferase. This motif is conserved in organisms from bacteria to higher eukaryotes. The Saccharomyces cerevisiae RFs, mitochondrial Mrf1p and cytoplasmic Sup45p (eRF1), have sequence similarities to the bacterial RFs, including the potential site of glutamine methylation in the GGQ motif. A computational analysis revealed two yeast proteins, Mtq1p and Mtq2p, that have strong sequence similarity to PrmC. Mass spectrometric analysis demonstrated that Mtq1p and Mtq2p methylate Mrf1p and Sup45p, respectively, in vivo. A tryptic peptide of Mrf1p, GGQHVNTTD-SAVR, containing the GGQ motif was found to be ϳ50% methylated at the glutamine residue in the normal strain but completely unmodified in the peptide from mtq1-⌬. Moreover, Mtq1p methyltransferase activity was observed in an in vitro assay. In similar experiments, it was determined that Mtq2p methylates Sup45p Chem. 280, 2439 -2445). Analysis of the deletion mutants showed that although mtq1-⌬ had only moderate growth defects on nonfermentable carbon sources, the mtq2-⌬ had multiple phenotypes, including cold sensitivity and sensitivity to translation fidelity antibiotics paromomycin and geneticin, to high salt and calcium concentrations, to polymyxin B, and to caffeine. Also, the mitochondrial mit ؊ mutation, cox2-V25, containing a premature stop mutation, was suppressed by mtq1-⌬. Most interestingly, the mtq2-⌬ was significantly more resistant to the anti-microtubule drugs thiabendazole and benomyl, suggesting that Mtq2p may also methylate certain microtubule-related proteins.Post-translational modification of proteins extends molecular structures beyond the limits imposed by the 20 encoded amino acids and, if reversible, allows a means of control and signaling. A wide range of prokaryotic and eukaryotic proteins are methylated post-translationally, including, for example, cytochrome c, ribosomal proteins, translation factors, and histones (1). The modifications occur by either N-methylation or carboxymethylation reactions, with the former reactions usually involving N-methylation of lysine, arginine, histidine, alanine, proline, glutamine, phenylalanine, asparagine, and methionine, whereas the latter reactions usually involving O-methylesterification of glutamic and aspartic acid. The enzymes catalyzing these methylation reactions generally use S-adenosylmethionine (AdoMet) 4 as the methyl donor to transfer the methyl group to the free amino group on the side chain of an amino acid residue (2). The extent of methylation can be complete or almost complete, as in the case of cytochrome c, or can be partial, as in case of ribosomal proteins. Once incorporated, the methyl groups do not appear to be removed from most proteins. However, reversible methylation of glutamic acid residues is involved in the chemotactic response of bacteria (3); also reversible methylation of the C subunit of the phosphoprotein phosphatase 2A (PP2A) at...

␣IIb␤3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail. Exogenous peptides designed to interact with the ␣IIb transmembrane (TM) domain activate single ␣IIb␤3 molecules in platelets by binding to the ␣IIb TM domain and causing separation of the ␣IIb␤3 TM domain heterodimer. Here we asked whether directly activating single ␣IIb␤3 molecules in platelets using the designed peptide anti-␣IIb TM also initiates ␣IIb␤3-mediated outside-in signaling by causing activation of ␤3-associated c-Src. Anti-␣IIb TM caused activation of ␤3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to ␣IIb␤3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual ␣IIb␤3 molecules can initiate ␣IIb␤3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-␣IIb TM resulted in the redistribution of ␣IIb␤3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered ␣IIb␤3. Thus, these studies indicate that not only is resting ␣IIb␤3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.␣IIb␤3 activation in platelets is followed by ␣IIb␤3 clustering (1) and ␣IIb␤3-mediated "outside-in" signal transduction (2) that is initiated by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the ␤3 cytosolic tail (CT) 3 (3-5). Activated c-Src then initiates an intracellular signaling cascade culminating in the reorganization of the platelet cytoskeleton, platelet spreading, and fibrin clot retraction, events important for efficient hemostasis in the hemodynamic environment of flowing blood (6).Recently, we reported studies of the interaction of c-Src with the ␤3 CT (5). We detected little to no interaction in unstimulated platelets, but following platelet stimulation with thrombin, c-Src associated with ␤3 in a time-dependent manner and underwent transient activation. We also found that the ␤3 CT binds to the c-Src SH3 domain at a site that is occupied by the linker connecting the c-Src SH2 and kinase domains in inactive c-Src. Following platelet activation, c-Src is "unlatched," allowing ␤3 CT binding to the SH3 domain when the linker binding site is vacated. However, the full catalytic activity of c-Src requires phosphorylation of Tyr 419 (numbered according to the UniProtKB/Swiss-Prot entry P12931 for human c-Src), located in a loop between the two lobes of the c-Src kinase domain (7). In platelets, this occurs by trans-autophosphorylation when ␣IIb␤3 clustering brings ␤3 CT-bound c-Src into proximity (3). Thus, ␣IIb␤3 clustering is an essential step in the outside-in signaling that follows ␣IIb␤3 activation.Transmembrane (TM) helix-helix interactions play an important role in maintaining ␣I...

Background: ␤3-bound c-Src initiates outside-in signaling in platelets. Results: After platelet stimulation, ␤3 binds to the c-Src SH3 domain at a site overlapping with its PPII helix binding site. Conclusion:The interaction of c-Src with ␤3 requires c-Src activation to vacate the PPII helix binding site. Significance: The mode of c-Src binding to ␤3 prevents c-Src-mediated signaling in circulating platelets.

Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.