GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4. Mutation of either Cys residue prevented association with DRMs. A hybrid protein containing the first 20 amino acid residues of GAP-43 fused to ␤-galactosidase was targeted to DRMs even more efficiently than GAP-43 itself. We conclude that tandem palmitoylated Cys residues can target GAP-43 to DRMs, defining a new signal for DRM targeting. We propose that tandem or closely spaced saturated fatty acyl chains partition into domains or "rafts" in the liquid-ordered phase, or a phase with similar properties, in cell membranes. These rafts are isolated as DRMs after detergent extraction. The brain-specific heterotrimeric G protein G o , which may be regulated by GAP-43 in vitro, was also enriched in DRMs from PC12 cells. Targeting of GAP-43 to rafts may function to facilitate signaling through G o . In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.GAP-43 (neuromodulin, B-50) is very abundant in developing and regenerating neurons and is also found in some neurons in the adult brain. It has been proposed to function in neuronal plasticity and the regulation of neurotransmitter release (1-5). GAP-43 binds calmodulin in low calcium and is a major substrate for protein kinase C. It has also been reported to modulate the activity of G o (the most abundant heterotrimeric G protein in growth cones) in vitro (6 -8), although the physiological relevance of this interaction remains controversial. Other evidence suggests a role in regulating neurite extension. Although neurite extension still occurs in mice lacking GAP-43, neuronal pathfinding is abnormal (9).Although GAP-43 is delivered to nerve terminals by fast axonal transport in association with vesicles, it does not contain a membrane-spanning domain (10, 11). Instead, GAP-43 binds membranes via palmitoylated Cys residues at positions 3 and 4 (12-14). GAP-43 has been detected in the Golgi region in neurons (15) and transfected fibroblasts (16). This pool of the protein is probably in the trans-Golgi network (TGN), 1 as its localization in transfected fibroblasts is not altered by brefeldin A (16). Targeting of GAP-43 to the TGN probably allows the protein to "hitchhike" to nerve terminals on the cytosolic surface of transport vesicles (16). Membrane proteins destined for axons and dendrites of neurons are probably sorted into different vesicles in the TGN, in the same way that apical and basolateral proteins are sorted in certain polarized epithelial cells (17). GAP-43 is expressed specifically in axonal growth cones in developing neurons (18), suggesting that it may be sorted into axonally directed vesicles in the TGN. As has...

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI-and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins. INTRODUCTIONIn contrast to transmembrane glycoproteins, surface molecules inserted into the plasma membrane via a glycosylphosphatidylinositol (GPI) 1 membrane anchor are confined to the outer leaflet of the plasma membrane and do not directly communicate with the cell interior (Low, 1989). However, several GPI-anchored proteins have been shown to be potent signal transducers, because their cross-linking leads to increased protein-tyrosine phosphorylation, calcium fluxes, gene expression, and cell activation and/or proliferation (Robinson, 1991). GPI-anchored proteins such as neurotrophic factor receptors transduce signals by ligand-induced interactions with transmembrane receptor protein-tyrosine kinases (PTKs) (Massague, 1996), whereas other GPI-anchored molecules such as CD87 (uPAR), CD16B (Fc␥RIIIB), and CD14 (lipopolysaccharide receptor) interact with integrins (Petty and Todd, 1996) and appear to signal via integrin-dependent pathways. For most other GPI-anchored proteins, signaling is presumed to require association with sphingolipid microdomains (Romagnoli and Bron, 1997;Stulnig et al., 1997), and their coprecipitation with Src family PTKs has been documented in hematopoietic , epithelial (ShenoyScaria et al., 1992), and neuronal (Zisch et al., 1995;Kunz et al., 1996) cells. The molecular nature of this indirect association between GPI-anchored receptors and Src kinases, however, remains unresolved. Sphingolipid microdomains are thought to consist of clusters of sphingolipids that achieve a liquid-ordered state in the presence of cholesterol (Ahmed et al., 1997;Schroeder et al., 1998) and resist solubilization by nonionic detergents. Such sphingolipid microdomain...

We had previously reported that splice isoforms of tenascin-C containing the extra-domain C are virtually absent in normal adult tissues but are highly abundant in high-grade astrocytomas, with a prominent peri-vascular pattern of expression. We now report that the extra-domain C of tenascin-C is strongly expressed in the majority of lung cancers, with a vascular and stromal pattern of expression. Using antibody phage technology, we have generated a human monoclonal antibody (G11), with a dissociation constant K(D) = 4.2 nM for the human domain C. The G11 antibody, expressed in scFv and in mini-antibody (SIP) format, as well as a scFv-interleukin-2 fusion protein, was then characterized in quantitative biodistribution studies using mice grafted subcutaneously with U87 gliomas, revealing a selective tumor uptake, with tumor/blood ratios up to 11.8:1 at 24 h. A radioiodinated preparation of SIP(G11) was also investigated in a double tracer study using an orthotopic rat glioma model, confirming the antibody's ability to preferentially localize at the tumor site, with tumor/brain ratios superior to the ones observed with (18)F-fluorodeoxyglucose. These tumor-targeting properties, together with the strong immunohistochemical staining of human tumor sections, indicate that the G11 antibody may be used as a portable targeting moiety for the selective delivery of imaging and therapeutic agents to gliomas and lung tumors.

Targeting the SDF-1-CXCR4 axis through CD26/DPP-4 inhibition increased the intragraft number of progenitor cells contributing to the recovery from ischaemia-reperfusion lung injury. Stabilization of endogenous SDF-1 is achievable and may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept.