Notch signaling is controlled by ligand binding, which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane-proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows ␥-secretase to execute a second cleavage within the Notch transmembrane domain, which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/tumor necrosis factor ␣-converting enzyme, plays an essential role in executing ligand-induced extracellular cleavage at site 2 (S2) in cells and localizes this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Because cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases has important implications for understanding Notch activation in normal and cancer cells.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.

Phosphatidic acid (PA), an intriguing phospholipid that is rapidly produced during receptor-stimulated breakdown of phosphoinositides, has often been proposed to function as a Ca2+ ionophore in activated cells. The PA-ionophore hypothesis is supported by the fact that exogenously applied PA stimulates Ca2+ uptake in various cells and can evoke Ca2+-mediated physiological responses, but it is not known whether PA accumulation affects cytoplasmic free Ca2+ concentration ([Ca2+]i). Here we report that PA elicits a transient rise in [Ca2+]i in cultured cells, not by stimulating Ca2+ influx, but, surprisingly, by releasing Ca2+ from intracellular stores. We further show that PA evokes growth factor-like effects in that it raises cytoplasmic pH, induces expression of the c-fos and c-myc proto-oncogenes and stimulates DNA synthesis. Our results indicate that, unlike an ionophore, PA acts by triggering the hydrolysis of phosphoinositides, with consequent formation of second messengers such as inositol trisphosphate signalling Cai2+ release. Furthermore, our data strengthen the notion that any Ca2+-mobilizing stimulus acting through phospholipase C may ultimately function as a growth factor.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct Gprotein-coupled receptors to evoke similar responses, including Ca# + mobilization, adenylate cyclase inhibition, and mitogenactivated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1\Edg-2) is similar to an orphan G icoupled receptor termed Edg-1. Here we show that expression of

Rap1 is a small GTPase regulating cell-cell adhesion, cell-matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1-ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.The mitogen-activated protein (MAP) 1 kinases are highly conserved serine/threonine protein kinases that are activated by diverse extracellular stimuli and mediate a wide variety of cellular responses (1-3). The p42/p44 MAP kinases, also named extracellular-regulated kinases (ERKs), function in a signaling cascade from the plasma membrane to the nucleus that controls cell cycle progression and differentiation and, furthermore, plays a key role in oncogenic transformation (1-3). Mitogens such as epidermal growth factor (EGF) and lysophosphatidic acid (LPA) trigger the MAP kinase cascade through activation of Ras via recruitment of the guanine nucleotide exchange factor Sos. Protein kinase C (PKC)-activating phorbol ester can also trigger the Ras-MAP kinase pathway, but the mechanism is distinct from that initiated by cell-surface receptors and presumably involves decreased Ras-GAP activity (4, 5). Active Ras binds to and activates the protein kinase c-Raf-1(referred to as Raf). Raf then phosphorylates and thereby activates the cytosolic MAP kinase kinases, MEK1 and MEK2, which in turn activate the p42/p44 MAP kinases (ERK1 and ERK2) by dual phosphorylation on threonine and tyrosine. Whereas Ras-Raf interaction takes place at the plasma membrane, it remains unclear where MEK activation occurs, at the membrane or in the cytoplasm. Activated MEK remains in the cytoplasm (6, 7), whereas activated MAP kinases can undergo translocation to the nucleus where they modulate gene expression through phosphorylation of transcription factors (1-3, 6, 8 -12).Recent evidence points to an essential, though poorly understood, role of receptor endocytosis in the activation of MAP kinase (13-19). Receptor endocytosis is regulated by dynamin, a 100 kDa GTPase that is targeted to clathrin-coated pits where it oligomerizes around the neck of budding vesicles (reviewed in Ref. 20), although its precise mode of action remains to be elucidated (Refs. 21 and 22, and references therein). Dynamin with a point mutation in the nucleotide-binding site (K44A) interferes with the function of endogenous dynami...

Abstract. We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAbl08), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a eDNA encoding the full-length human EGF receptor gene, and fully responsive to EGE The binding to saturation of mAbl08 to the surface of these cells at 4°C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGE The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37°C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 106 M-~-s -~) than did low-affinity receptor (2.5 × 105 M-Ls-~), whereas the off-rate constants, measured at 4°C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors.At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAbl08 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca 2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAbl08 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication. (ii) the ZO-1 'scaffold' protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of alpha/beta-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds alpha-tubulin equally well as beta-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.