Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/ gelatin-binding region between domains FNI 6 and FNI 9 . INTRODUCTIONFibrillins are extracellular matrix components with important functions in elastic and nonelastic tissues including blood vessels, bone, and the eye. Fibrillins, together with the latent transforming growth factor-␤ (TGF-␤)-binding proteins (LTBPs), constitute the fibrillin-LTBP family of proteins (Hubmacher et al., 2006). Fibrillins are ϳ350 kDa in size, are disulfide-rich and glycosylated, and have a characteristic modular structure. The three human fibrillinsfibrillin-1, -2, and -3 -are encoded by different genes and are conserved at the amino acid level both in relation to one another and among species. Fibrillins are mainly composed of tandem arrays of calcium-binding epidermal growth factor-like domains interspersed with TGF-␤-binding protein domains (TB/8-Cys) and hybrid domains Hubmacher et al., 2006). Mutations in fibrillins give rise to the so-called fibrillinopathies, which include Marfan syndrome and autosomal dominant Weill-Marchesani syndrome, both caused by mutations in fibrillin-1, and Beal's syndrome, caused by mutations in fibrillin-2 (Robinson et al., 2006).High-molecular-weight, multiprotein assemblies called microfibrils are the functional units of fibrillins, which serve as a scaffold for the biogenesis of elastic fibers, confer structural integrity to individual organ systems, regulate growth factor signaling of TGF-␤-bone morphogenic protein (BMP) superfamily members and provide limited elasticity to tissues (Kielty et al., 2002;Charbonneau et al., 2004;Ramirez and Dietz, 2007). Extracted microfibrils from cell culture or tissues display a typical bead-on-a-string ultrastructure, having a 50 -55-nm periodicity when analyzed by ele...

Matrix metalloproteases regulate both physiological and pathological events by processing matrix proteins and growth factors. ADAMTS1 in particular is required for normal ovulation and renal function and has been shown to modulate angiogenesis. Here we report that TSP1 and 2 are substrates of ADAMTS1. Using a combination of mass spectrometry and Edman degradation, we mapped the cleavage sites and characterized the biological relevance of these processing events. ADAMTS1 cleavage mediates the release of polypeptides from the trimeric structure of both TSP1 and 2 generating a pool of antiangiogenic fragments from matrix-bound thrombospondin. Using neo-epitope antibodies we confirmed that processing occurs during wound healing of wild-type mice. However, TSP1 proteolysis is decreased or absent in ADAMTS1 null mice; this is associated with delayed wound closure and increased angiogenic response. Finally, TSP1À/À endothelial cells revealed that the antiangiogenic response mediated by ADAMTS1 is greatly dependent on TSP1. These findings have unraveled a mechanistic explanation for the angiostatic functions attributed to ADAMTS1 and demonstrated in vivo processing of TSP1 under situations of tissue repair.

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRP␣ (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1-and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction. NO preserves vascular health by minimizing the adhesion of inflammatory cells to the vessel wall, limiting platelet activation, and increasing blood vessel diameter and blood flow by relaxing vascular smooth muscle cells (VSMC).3 These actions of NO are mediated by activating soluble isoforms of guanylate cyclase (sGC) to increase cGMP levels, resulting in downstream activation of cGMP-dependent protein kinases and ion channels (1).Physiological NO/cGMP signaling is limited by several phosphodiesterases that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein that is produced by vascular and inflammatory cells that regulates cellular behavior by engaging several cell surface receptors. Recently we reported that TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and VSMC (2, 3). TSP1 also plays a role in promoting platelet thrombus formation and hemostasis by antagonizing the antithrombotic activity of NO (4). In all of these vascular cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated fluxes in cGMP by engaging its receptor CD47 (5). Nanomolar concentrations of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated uptake of myristate into vascular cells (6). In vivo, mice lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater increases in regional blood flow in response to a NO challenge than wild type controls (4). After an ischemic insult, the absence of TSP1 o...

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin α4β1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of α4β1 integrin, and TSP1 inhibited interaction of activated α4β1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The α4β1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the α4β1 integrin–dependent activities of TSP1. The β1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.