Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain

135

104

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...

“…GAP-43 is enriched in DRMs isolated from rat brain (40). To determine whether GAP-43 is present in PC12 cell DRMs, solubilized DRMs were incubated with anti-GAP-43 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Association of GAP-43 with Detergent-resistant Membranes Requires Two Palmitoylated Cysteine Residues

Arni

Keilbaugh

Ostermeyer

et al. 1998

135

104

“…Preparation of TIF from Rat Brain-TIF was prepared as described previously with slight modification (13). All procedures were carried out on ice or at 4°C, unless otherwise mentioned.…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE, Western blotting using ECL system (Amersham Pharmacia Biotech), and protein determination were performed as described (13). Anti-Thy-1 antibodies were obtained from Cedarlane and Sigma.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

Funatsu

Miyata²,

Kumanogoh

et al. 1999

Self Cite

109

100

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-weekold rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

“…The palmitoylation motif of GAP-43 can mediate lipid raft association for protein trafficking to the axonal membrane. Indeed, previous studies have found that GAP-43 is enriched in DIGs from brain homogenates (31) and is transported to axons on vesicles derived from the secretory pathway (32). On the other hand, signals within the body of PSD-95 working together with palmitoylation may mediate its association with dendritic targeting vesicles and its exclusion from rafts.…”

Section: Targeting Of Peripheral Membrane Proteins In Neurons 44987mentioning

confidence: 96%

Polarized Targeting of Peripheral Membrane Proteins in Neurons

El-Husseini¹,

Craven²,

Brock

et al. 2001

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growthassociated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.Proper neuronal function requires selective protein targeting to specialized cellular and plasma membrane domains including the nerve terminal, node of Ranvier, axon hillock, and postsynaptic density. An early step in this targeting decision tree involves a polarized sorting of proteins to either dendritic (postsynaptic) or axonal (presynaptic) domains. However, the mechanisms by which neurons target specific proteins to dendrites versus axons are poorly understood. Better characterized is protein sorting to apical versus basolateral plasma membranes in polarized epithelial cells, which share certain features with axonal versus dendritic targeting in neurons (1, 2). That is, short cytosolic C-terminal protein-sorting motifs are one route for both dendritic and basolateral targeting (2), whereas specialized lipid rafts can mediate both axonal and apical sorting of certain transmembrane and glycosylphosphatidylinositol-anchored membrane proteins (3).The concept of specialized lipid rafts mediating polarized protein targeting emerged from observations that apical and basolateral cell membranes have different lipid compositions. Apical membranes are enriched in sphingolipids that aggregate with cholesterol to form packed raft-like domains within the fluid membrane bilayer. These rafts are insoluble in nonionic detergents and, hence, are termed detergent-insoluble glycolipid-enriched complexes (DIGs).1 These complexes form in the trans-Golgi network and incorporate certain transmembrane, GPI-anchored, and dually acylated proteins, which are then targeted to the apical plasma membrane (4, 5). The inhibition of DIG formation by sphingolipid or cholesterol depletion disrupts this apical/axonal sorting pathway (3, 6, 7). However, the polarized targeting of cytosolic proteins via DIGs has not been explored.Postsynaptic density-95 (PSD-9...