B-50/GAP43 is localized at the cytoplasmic side of the plasma membrane in developing and adult rat pyramidal tract

Gorgels, T. G. M. F.; Campagne, Menno van Lookeren; Oestreicher, A.B.; Gribnau, A.A.M.; Gispen, Willem Hendrik

doi:10.1523/jneurosci.09-11-03861.1989

Cited by 96 publications

(58 citation statements)

References 39 publications

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“…GAP-43 (also known as neuromodulin, B-50, F1, and P57) is enriched in the growth cone membranes of elongating axons (5)(6)(7)(8)(9) and is proposed to function in neuronal plasticity and the regulation of neurotransmitter release (10 -13). GAP-43 is initially synthesized as a soluble protein, which subsequently passes through the secretory pathway, and becomes membrane-bound post-translationally.…”

mentioning

confidence: 99%

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Liang

Neubert

et al. 2002

Journal of Biological Chemistry

101

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GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.Covalent modification by acetylation or fatty acylation occurs on a variety of viral and cellular proteins (1). N-terminal acetylation is one of the most common protein modifications, occurring on ϳ85% of eukaryotic proteins (2). The amino acid residue adjacent to the amino-terminal methionine residue determines whether the N-terminal methionine is retained or removed before acetylation (2). Proteins that contain the Nterminal sequence MGXXX(S/T) undergo a different set of modifications. The initiating Met is removed, and myristate is added to the N-terminal glycine. The requirement for glycine at the N terminus is absolute for N-myristoylation to occur.In contrast to N-terminal acetylation and myristoylation, which occur co-translationally, palmitoylation is a post-translational lipid modification. Nearly all palmitoylated proteins are S-acylated by attachment of palmitate via a thioester linkage to the sulfhydryl group of cysteine. Exceptions include adenylate cyclase toxin from Bordetella pertussis, which is modified by amide-linked palmitoylation on the ⑀-amino group of lysine residues (3) and human sonic hedgehog, which is palmitoylated through an amide linkage to the N-terminal cysteine (4). Unlike myristoylation, the enzymology of palmitoylation reactions is poorly understood. Palmitoylacyl transferases have not been thoroughly purified and characterized. Moreover, no study has directly examined the nature of thioester-linked palmitoylation in vivo at a molecular level. All of the studies on thioester-linked palmitoylation are based on incorporation of radioactive palmitate. The stoichiometry of palmitoylation as well as the molecular identity of the attached fatty acid moiety (palmitate a...

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mentioning

confidence: 99%

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Liang

Neubert

et al. 2002

Journal of Biological Chemistry

101

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“…The immunostaining pat terns for CGRP and B-50 that were observed after 6 CGRP antibodies in bones with an implant revealed weeks were morphologically similar to those for the many varicose fibres in almost all of the remodeling growth cones of the regenerating sciatic nerve (52) cavities. In the sampling areas, 23,4 ± 5.2 remodeling and those reported in the developing pyramidal tract cavities were found, and they contained 483 ± 79.5 (10,11,28). Althoimh the exact nature of these strucneural varicosities (a mean of 21 varicosities per cav ity).…”

Section: Discussionmentioning

confidence: 78%

“…The specificity of the antibodies to ,CGRP (5) and B-50 (10,11,39) has been tested previously. The specificity of the antisera was determined with use of the conven tional method of substituting normal rabbit preimmune or mouse serum for the primary antiserum and omitting the primary antise rum step.…”

Section: Iniinunocytocheniistrymentioning

confidence: 99%

“…thus, the antibody may be of use to demonstrate axHowever, it also seems very plausible that CGRP plays onal sprouting (11,49,53). The purpose of this study was to describe the changes in the pattern of sensory innervation in bone after insertion of an implant.…”

mentioning

confidence: 99%

“…This antibody has been applied to detect vascularization or bone remodeling, and pain and pro prioception (2, 3,19,26), outgrowing nerve fibres (48,52,53). A high concen tration of B-50/GAP-43 has been found in axonal The present study focussed on the changes in the growth cones during embryonic (37,50) and post innervation pattern of sensory fibres associated with natal development (10,11,39). The expression of Bbone remodeling after insertion of an implant.…”

mentioning

confidence: 99%

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Changes in innervation of long bones after insertion of an implant: Immunocytochemical study in goats with antibodies to calcitonin gene‐related peptide and B‐50/GAP‐43

Buma¹,

Elmans²,

Oestreicher

1995

Journal Orthopaedic Research

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Sum m ary: In this study, we describe the distribution of fibres th a t co n tain calcitonin g e n e -re la te d p e p tid e in norm al bones and in bones th a t are rem odeling a fter insertion of an im plant. W ith ro u tin e histology and a n ti bodies to calcitonin gen e-related peptide, sm all neural and free-ru n n in g fibres staining positively lo r c alcito nin gene-related p ep tid e w ere found in the p erio steu m , en d o steu m , and cortical bone of the tibia in the goat. In m any cases, th e free-running fibres w ere associated with b lood vessels th a t e n te re d the b o n e th ro u g h V olkm a n n 's canals. T he endosteal b lood supply was d estro y ed as a result of in sertio n of th e im plant. T h e n ecro tic b o n e was no longer innervated, as show n by th e lack of staining for th e antibodies. A t 6 w eeks, a re p a ir p h ase sta rte d with revascularization and rem o d elin g of the necrotic en d o steal bone. D u rin g this re p a ir phase, th e re was increased innervation with fibres containing calcitonin g en e-related p e p tid e in the rem o d elin g cavities at the interface betw een living and necrotic bone, T hese fibres en d ed blindly, w ith m any large varicosities, an d could be d em o n strated by im m unostaining with m onoclonal antibodies to B -50/grow th associated p ro tein -4 3 , an an tib o d y to outgrow ing n eu ro n al fibres. T he correlative occurrence b e tw e e n extensive sp ro u tin g o f fibres containing calcitonin g en e-related p ep tid e and the rem odeling of necrotic en d o steal bone suggests th a t s e n sory fibres with calcitonin g en e-related p ep tid e have a regulatory role in the control of angiogenesis o r o f b o n e rem odeling associated with th e insertion of an im plant, or with both processes.il i « i r » É ) i n n i r r r o r i i w i * -Î

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Postnatal development of GAP-43 immunoreactivity in the auditory brainstem of the rat

1997

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Extensive data link the growth associated protein GAP-43 to axonal elongation and synapse formation during development and in plastic responses of nervous tissue. We have studied the changing levels of GAP-43 expression in the auditory brainstem nuclei of the developing rat by applying immunocytochemical techniques. By the first postnatal day (P1), GAP-43 was expressed at high concentrations in all subdivisions of the cochlear nuclear complex and the superior olivary complex. At this stage, neuropil structures recognized by the antibody did not show any varicosities on cellular processes in all these regions. By P8, the texture of the stain has turned markedly more granular, a pattern likely to reflect the formation of presynaptic endings. A predominantly granular distribution of GAP-43 has developed by P12. At that time, the staining intensity is markedly reduced compared to the levels of the newborn. By P16, the auditory brainstem nuclei have lost most of their GAP-43 immunoreactivity, but a distinct level of staining persisted into adulthood in all of them. This staining was restricted to boutons, which are thought to be presynaptic terminals. We conclude that a moderate but apparently relevant potential for plasticity is retained in these auditory structures. Should the patterns of neural signals, mediated by the inner ear, change during adulthood, the central structures appear to be able to respond with the formation of altered synaptic connectivity.

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B-50/GAP43 is localized at the cytoplasmic side of the plasma membrane in developing and adult rat pyramidal tract

Cited by 96 publications

References 39 publications

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

Changes in innervation of long bones after insertion of an implant: Immunocytochemical study in goats with antibodies to calcitonin gene‐related peptide and B‐50/GAP‐43

Postnatal development of GAP-43 immunoreactivity in the auditory brainstem of the rat

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