Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear.In this report, we show that the palmitate of SNAP-25 is rapidly turned over in Pci 2 cells, with a half-life of -~3h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys 85'88 or Cys9092 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation. Key Words: Synaptic proteins-SNAP-25-Palmitoylation-Membrane association-Mutagenesis-Protein turnover. J. Neurochem. 69, 1864Neurochem. 69, -1869Neurochem. 69, (1997.The synaptosomal associated protein of 25 kDa, SNAP-25, is a member ofthe synaptic vesicle docking! fusion complex that includes syntaxin, synaptobrevin (VAMP), and synaptotagmin (for reviews, see Bennett and Scheller, 1994;Ferro-Novick and Jahn, 1994;Rothman and Warren, 1994; Sudhof, 1995). It has been shown to interact with both syntaxin and synaptobrevin by in vitro binding assays (Chapman et al., 1994; Pevsner et al., 1994; McMahon and Sudhof, 1995). Analysis of cDNA clones of SNAP-25 from mouse (Oyler et al., 1989), chicken (Catsicas et al., 1991), and Drosophila and Torpedo showed that the protein is highly conserved through evolution and is expressed specifically in the nervous systems. SNAP-25 plays an important role in synaptic vesicle docking/fusion. Specific cleavage of the protein by botulinum neurotoxins A and E blocks neurotransmitter release at neuromuscular junctions (Biasi et al., 1993;Schiavo et a!., 1993;Binz et al., 1994). More recently, SNAP-25 homologues from yeast and nonneuronal tissues were cloned, which showed high degrees of homology in the important functional domains (Brennwald et al., 1994;Ravichandran et al., 1996). These results strongly suggest that SNAP-25 and its homologues play fundamental roles in vesicular transport.SNAP-25 is localized primarily to the membrane fractions in both developing and adult brains (Oyier et a!., 1989). It is one of the major palmitoylated proteins found in neurons (Hess et a!., 1992), and this posttranslational modification may play an important role in modulating the physiological functions of the protein. Deletion of a 12-amino acid sequence containing the four Cys residues of SNAP-25 abolished palmitoylation and membrane association of the protein, suggesting that paimitoylation may be required for membrane association (Veit et al., 1996). Two SNAP-25 isoform...

Abstract. MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KC1, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca 2÷ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KC1 increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth.

Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein which is implicated in neuronal growth and regeneration. It is concentrated in neuronal growth cones and associates with membranes through the palmitoylation of the N-terminal peptide MLCCMRRTK at Cys-3 and Cys-4. In the present study, we have identified critical amino acid residues required for palmitoylation and membrane association of neuromodulin in vivo. Several neuromodulin constructs with point mutations were tested for membrane association and palmitoylation. Wild-type neuromodulin expressed in COS-7 cells incorporated [3H]palmitic acid, whereas a mutant in which both Cys-3 and Cys-4 were substituted with glycine was not palmitoylated in vivo. Mutant proteins in which either Cys-3 or Cys-4 was substituted with leucine incorporated 75% and 25% of [3H]palmitic acid, respectively, compared to wild-type neuromodulin. The relative distribution of mutant neuromodulins expressed in COS-7 cells was quantitated by immunoblot analysis of the membrane and cytosolic fractions. There was a general correlation between membrane association of mutant neuromodulins and the extent to which they were palmitoylated in vivo. Additional point mutations in the acylation domain of neuromodulin indicated that a short hydrophobic amino acid sequence N-terminal to Cys-4 may be required for optimal palmitoylation and membrane association. We conclude that Cys-4 is critical for the palmitoylation and membrane association of neuromodulin.

Background Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. Methods and Results Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes of Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules (CAMs) such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. Conclusions Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, lipid raft clustering and CAM trafficking modulate angiogenic potential, and the perturbation of CD82-ganglioside-CD44 signaling attenuates angiogenesis.