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 β-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.

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.

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 ¹⁸F-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.

Lipoarabinomannans (LAMs) are major Ags of the mycobacterial cell envelope where they apparently insert through a glycosylphosphatidylinositol (GPI) anchoring structure. LAMs induce host macrophages to secrete TNF-α, IL-1, and IL-6 and inhibit T cell proliferative responses. The mechanisms by which LAMs mediate these effects remain poorly understood. We show that LAMs were efficiently inserted into the plasma membranes of human and murine lymphomonocytic cells through their GPI anchor. Prior deacylation of LAMs abrogated this event. Phosphatidylinositol hexamannoside (PIM6), the GPI anchor of all LAMs, competitively inhibited LAM insertion. Deacylated PIM6 was not inhibitory. The hexamannoside glycan of PIM6 appears to be important for LAM insertion, since phosphatidylinositol from soybean, lacking the glycan core, was not as efficient an inhibitor. Interaction of LAM with target cells was influenced by the gel/fluid phase distribution of membrane lipids, suggesting a direct interaction of the LAM-GPI anchor with the membrane bilayer. The inserted LAMs were mobile in the plane of the membrane and interfered with Ab-mediated mobilization of the GPI-anchored Thy-1 molecules. Further, LAMs were preferentially incorporated into isolated plasma membrane vesicles enriched in Thy-1. Our results strongly suggest that 1) interaction of LAMs with host lymphomonocytic cells is mediated through a preferential integration of LAM-GPI anchor into specialized plasma membrane domains enriched in endogenous GPI-anchored molecules, and 2) both the acyl chains and the mannoside core glycan of the LAM-GPI anchor contribute to the specificity of integration.