Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Doherty, Patrick; Rowett, L H; Moore, Stephen E.; Mann, Derek A.; Walsh, Frank S.

doi:10.1016/0896-6273(91)90360-c

Cited by 174 publications

(115 citation statements)

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“…Ectopic expression of cytNCAM-140 inhibits NCAM-stimulated neurite outgrowth NCAM has been shown to stimulate neurite outgrowth in both neurons and PC12 cells (Doherty et al, 1990(Doherty et al, , 1991a. Thus, the latter cells are regarded as a convenient and reliable model for the study of NCAM-mediated neuritogenesis.…”

Section: Resultsmentioning

confidence: 99%

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

Kolkova

Pedersen

Berezin

et al. 2000

Journal of Neurochemistry

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Abstract:The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cyt-NCAM-140) and of the 180-kDa NCAM isoform (cyt-NCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839 -843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu 840 and Lys 842 with Ala abrogated the effect of the construct, assigning a critical role to these two residues. Key Words: NCAM-Cytoplasmic domain-Fibroblasts-Neurite outgrowth.

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Section: Resultsmentioning

confidence: 99%

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

Kolkova

Pedersen

Berezin

et al. 2000

Journal of Neurochemistry

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“…We have previously demonstrated that neurons extend longer neurites when cultured on a monolayer of transfected 3T3 cells that express physiological levels of N-cadherin, as compared with untransfected 3T3 cells (8,28,29). This response is driven by the homophilic binding of N-cadherin in the neurons to the transfected N-cadherin present in the 3T3 cell monolayers, and this model is one of the few quantitative assays that measures a biological response to physiological levels of N-cadherin.…”

mentioning

confidence: 99%

“…The first extracellular domain (ECD1) 1 of these cadherins contains an evolutionarily conserved HisAla-Val (HAV) motif (6,18), and several lines of evidence suggest that this sequence is critical for function. In this context, synthetic peptides containing the HAV motif (for example, N-Ac-LRAHAVDING-NH 2 ) have been shown to be capable of inhibiting cadherin-dependent biological processes, such as myoblast fusion (19), neurite outgrowth (8), and embryo compaction (18). Furthermore, antibodies directed against the HAV sequence are also capable of disrupting cadherin-dependent cell adhesion (5,20,21).…”

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confidence: 99%

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A Novel Family of Cyclic Peptide Antagonists Suggests That N-cadherin Specificity Is Determined by Amino Acids That Flank the HAV Motif

Williams

Gour

et al. 2000

Journal of Biological Chemistry

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142

132

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The classical cadherins (e.g. N-, E-, and P-cadherin) are well established homophilic adhesion molecules; however, the mechanism that governs cadherin specificity remains contentious. The classical cadherins contain an evolutionarily conserved His-Ala-Val (HAV) sequence, and linear peptides harboring this motif are capable of inhibiting a variety of cadherin-dependent processes. We now demonstrate that short cyclic HAV peptides can inhibit N-cadherin function. Interestingly, the nature of the amino acids that flank the HAV motif determine both the activity and specificity of the peptides. For example, when the HAV motif is flanked by a single aspartic acid, which mimics the natural HAVD sequence of N-cadherin, the peptide becomes a much more effective inhibitor of N-cadherin function. In contrast, when the HAV motif is flanked by a single serine, which mimics the natural HAVS sequence of E-cadherin, it loses its ability to inhibit the N-cadherin response. Our results demonstrate that subtle changes in the amino acids that flank the HAV motif can account for cadherin specificity and that small cyclic peptides can inhibit cadherin function. An emerging role for cadherins in a number of pathological processes suggests that the cyclic peptides reported in this study might be developed as therapeutic agents.The classical cadherins are a family of calcium-binding integral membrane glycoproteins that can account for the ability of cells to segregate from each other during development (1-3). In this context cadherins have been shown to regulate epithelial (4), endothelial (5), and neural (6 -8) cell adhesion. N-cadherin is predominantly expressed on neural, endothelial, and invasive cancer cells, whereas E-cadherin is predominantly expressed by epithelial cells (9 -11). The classical cadherins generally promote cell adhesion by interacting with one another in a homophilic manner (12, 13). In addition, the classical cadherins perform numerous other biological functions (1). For example, E-cadherin has been shown to be capable of acting as a tumor suppressor (14,15), and N-cadherin promotes neurite outgrowth (8).The classical cadherins are composed of five extracellular domains, a single transmembrane domain, and two cytoplasmic domains (1,16,17). The first extracellular domain (ECD1) 1 of these cadherins contains an evolutionarily conserved HisAla-Val (HAV) motif (6, 18), and several lines of evidence suggest that this sequence is critical for function. In this context, synthetic peptides containing the HAV motif (for example, N-Ac-LRAHAVDING-NH 2 ) have been shown to be capable of inhibiting cadherin-dependent biological processes, such as myoblast fusion (19), neurite outgrowth (8), and embryo compaction (18). Furthermore, antibodies directed against the HAV sequence are also capable of disrupting cadherin-dependent cell adhesion (5,20,21).Crystal studies have demonstrated that N-cadherin ECD1 monomers will form two types of dimeric structure, one that might reflect a trans adhesion interface, and one that might reflec...

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“…Furthermore, cells potentially possess distinct repertoires of glycosyltransferases that could synthesize P r F molecules with diverse carbohydrate structures (Wiley and Skehel, 1987;BrandIey et al, 1990;Doherty et al, 1991;Key and Akeson, 199 1;Takeuchi and Kobata, 1991). This raises the possibility that the propagation of a particular prion isolate results from synthesis of nascent PrPsc moiecules within a restricted subset of cells.…”

Section: Prion Diversitymentioning

confidence: 99%

The Neurochemistry of Prion Diseases

DeArmond

Prusiner

1993

Journal of Neurochemistry

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Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Cited by 174 publications

References 45 publications

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

A Novel Family of Cyclic Peptide Antagonists Suggests That N-cadherin Specificity Is Determined by Amino Acids That Flank the HAV Motif

The Neurochemistry of Prion Diseases

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