SLAIN2’s interactions with multiple different microtubule plus end–tracking proteins stimulate processive microtubule polymerization and ensure proper microtubule organization.

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligandbinding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/ VEGFR-2 ECD complex derived from smallangle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation. (Blood. 2012;119(7):1781-1788) IntroductionA plethora of growth factors, such as angiopoietins, VEGF family ligands, platelet-derived growth factors, fibroblast growth factors, and hepatocyte growth factors regulate blood and lymph vessel formation and homeostasis (reviewed in Cao 1 ). VEGFs represent a large family of ligands: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF, which bind to and activate in a combinatorial fashion 3 type V receptor tyrosine kinases (RTKs), VEGFR-1, VEGFR-2, and VEGFR-3, which give rise to highly specific signal output. In mammals, VEGF-A signaling through VEGFR-2 is the major angiogenic signaling pathway, but VEGF-C plays essential, and in some cases complementary, roles in the activation of this receptor (reviewed in Grünewald et al 2 ). The mechanism by which VEGFRs are activated is not understood in molecular detail, but clearly represents one of the many variations of RTK activation. In general, signaling by RTKs requires ligand-mediated dimerization with precise positioning of receptor subunits in active dimers. Dimeric ligand/receptor complexes subsequently initiate transmembrane signaling, resulting in the activation of the intracellular tyrosine kinase domains. 3,4 Active VEGFRs instigate cell signaling and promote endothelial cell migration and proliferation, as well as vessel fenestration and permeabilization. 5,6 The extracellular domain (ECD) of VEGFRs consists of 7 Ig-homology domains. The first 3 domains mediate ligand binding, 7,8 whereas the membrane proximal domains are involved in ligand-induced receptor dimerization. 7,9 Homotypic receptor i...

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membranedistal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.Peroxisomes are single membrane-bound organelles that carry out a variety of metabolic processes. In addition to the degradation of H 2 O 2 , the ␤-oxidation of very long chain or branched chain fatty acids and the synthesis of ether lipids are performed in these subcellular compartments (1, 2). The biogenesis of peroxisomes, including their formation and proliferation, as well as the degradation of peroxisomes are highly dynamic processes that are adapted to metabolic needs (3). Defects in peroxisome biogenesis cause a number of severe inherited diseases, which are collectively referred to as peroxisome biogenesis disorders (4, 5). Studies in yeast and analysis of patients affected by these disorders have led to the identification of specific proteins involved in peroxisomal formation and maintenance (6). Fifteen such proteins, which are named peroxins, are currently known in humans and the corresponding genes (PEX genes) are highly conserved throughout the eukaryotic kingdom (7,8).All matrix proteins and most membrane proteins are imported post-translationally into peroxisomes. The machinery of peroxins that mediates the import of matrix proteins bearing a peroxisomal targeting signal is far better understood than the machinery that mediates the recognition and import of membrane proteins (9, 10). The peroxins PEX3, 5 PEX16, and PEX19 are known to be essential for peroxisomal membrane biogenesis as a loss of any of these proteins leads to the complete absence of detectable peroxisomal membrane structures (11). However, de novo formation of peroxisomes was observed in cells deficient for each of these peroxins upon complementation with the wild type gene, raising an intriguing question about the origin of the peroxisomal membrane (11-15). The endoplasmic reticulum membrane as the obvious source was disputed for a long time as several studies indicate that this process does not involve the classical coat protein I-and coat protein II-dependent pathways (16 -18). Recently, new evidence for an involvement of the endoplasmic reticulum as a peroxisomal precursor has been reported in ...

EBs, key microtubule (MT) tip–tracking proteins, are elongated molecules with two interacting calponin homology (CH) domains, an arrangement reminiscent of MT- and actin-binding CH proteins. In addition, electrostatic interactions between the C-terminus of EBs and MTs drive the specificity of EBs for growing MT ends.

Microtubule plus-end tracking proteins (؉TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of ؉TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse ؉TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo-versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycinerich domains of either CLIP-170 or p150 glued or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.