The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21w has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchangepromoting protein acts reversibly and does not cause degradation of p211. It is inactive towards the a subunit of a heterotrimeric GTP-binding protein (G..) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchangepromoting), has the characteristics expected of a physiological activator of p21w in cellular growth-signal-transduction pathways.Members of the ras family of oncogenes encode Mr 21,000 proteins (p21) that bind and hydrolyze guanine nucleotides. The transforming potential of the ras protooncogenes can be activated by point mutations that either decrease the intrinsic GTPase activity of p21 or increase the rate with which p21 exchanges guanine nucleotides (1). Activated ras oncogenes have been found in up to 30% of human tumors (2). By analogy with other oncogene products, it has been speculated that p21 normally functions as a component of a growthsignal-transduction pathway. One would therefore expect it to interact with several distinct cellular proteins that act variously to impart signals to p21, to receive signals from p21, or to modulate signal transduction. A protein that activates the GTPase activity of p21 (GAP) has been identified (3); this may represent a downstream effector ofp21 action (4, 5). The nature of the upstream elements of the pathway that control p21 activity remains unknown: by analogy with the heterotrimeric GTP-binding proteins (G proteins) (6) it might be expected that stimulation of the activity of p21 would involve an increase of the rate at which it exchanges guanine nucleotide with soluble pools. Here we report the identification of an activity in a human tissue extract that promotes the exchange of guanine nucleotides on p21. This factor, termed REP (ras exchange-promoting), has properties consistent with its being a physiological activator of the ras proteins.MATERIALS AND METHODS Purification and Assay of Exchange Activity. Two normal human placentas were homogenized in a domestic blender with 1 liter of ice-cold 50 mM potassium phosphate buffer (pH 7.5) containing 1 mM EGTA, 0.3 M sucrose, 1 mM dithiothreitol (DTJ7), 10 gg of soybean trypsin inhibitor per ml, 10 ,Ag of leupeptin per ml, 10 ,ug of aprotinin per ml, and 10 mM benzamidine. Particulate matter was pelleted by centrifugation at 50...

Proteins encoded by ras genes have recently been reported to couple certain growth factor receptors to phospholipase C, the enzyme catalyzing phosphatidylinositol breakdown. To investigate this hypothesis, the normal and the transforming Ha-, Ki-, and N-ras genes were each transfected into Rat-i fibroblasts under the control of strong promoters.Several cell lines, both normal and transformed, were selected that expressed high levels of p2lr,. Phosphatidylinositol turnover was measured in these cells in response to a wide variety of peptide factors; bradykinin was found to have a greatly enhanced effect on the p21r overexpressors relative to the parental and control cells. Bradykinin receptor numbers were measured in these lines and found to be up to 40-fold higher in the p2lras overexpressors than in the parental cells. This was found to be the case for both normal and transforming forms of all three varieties of ras genes. Receptor number correlated well with the bradykinin-dependent phosphatidylinositol turnover response in all cases. These data indicate that the effects of p21r on cellular responses to the peptide hormone bradykinin are due to changes in receptor number rather than to direct coupling by p21r between the receptor and phospholipase C.The eukaryotic ras genes encode a family of proteins (M, 21,000) that appear to be important in control of cell growth. The acutely transforming Harvey and Kirsten murine sarcoma viruses contain activated mutants of Ha-and Ki-ras genes, and oncogenic forms of the Ha-and Ki-ras as well as of the related N-ras gene have been identified in a large number of human tumors (referred to as HRAS, KRAS, and NRAS in human gene nomenclature) (1-4). The mechanisms by which activated p2iras transforms cells and the ways in which normal p21ras may be involved in cellular growth control are currently unclear, although in recent years several clues have emerged. The ras proteins are peripheral membrane proteins (5) that bind and hydrolyze GTP; the activated proteins have reduced GTPase activity (6, 7). This points to a functional homology with members of the family of G proteins, GTP-binding proteins that are involved in the coupling of cell-surface receptor proteins to intracellular enzyme systems, most notably adenylate cyclase and cGMP phosphodiesterase (8). A limited sequence homology also exists between p2iras and the G proteins (9-12), so it is tempting to speculate that, despite their considerable differences, the ras proteins may also couple cell-surface receptors to intracellular enzymes. One enzyme known to be controlled by poorly characterized G proteins and to be of great importance in the regulation of cell growth is phospholipase C, the phosphodiesterase responsible for phosphatidylinositol (Ptdlns) breakdown (13).Some recent reports have presented evidence that p2lras may indeed be involved in the direct control of this enzyme (14, 15). The strategy used to study p21s-mediated linkage between receptors and phospholipase C has involved creation of cell lines ...

The products of the ras oncogenes (p2l) are ubiquitous membrane-associated proteins that bind guanine nucleotides and possess an intrinsic GTPase activity. Because of their functional homologies with regulatory guanine nucleotide-binding proteins, they are thought to be involved in the control of cellular proliferation as transducers of incoming growth signals. In an effort to identify proteins interacting with p2lr', we have used in vivo crosslinking techniques on Rat-1 fibroblasts and derived cell lines overexpressing p2l and immunoprecipitation with polyclonal anti-p21ra antibodies. Under those conditions, using the homobifunctional crosslinker dithiobis(succinimidyl propionate), a protein of Mr 60,000 (p60) is found to be associated with p2l ¶, and this association is enhanced by the treatment of quiescent cells with serum. Upon sedimentation ofdetergent extracts from crosslinked cells on sucrose gradients, a p21-p60 complex could be demonstrated with a Mr of 200,000-300,000. p60 does not appear to be related to pp60irC nor to the cytosolic GTPase activating protein that interacts with p2l to enhance its GTPase activity. The amount of p60 seems to be limiting relative to p2l1 in fibroblasts, since similar levels of p60 are immunoprecipitated from Rat-i cells and transfectants overexpressing Ha-, Ki-, and N-ras p21s; the same protein is also found to associate with p2iraS in numerous mammalian cell lines. The relevance of this component to the role of ras proteins in signal transduction is discussed.The mammalian Ha-, Ki-, and N-ras

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