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Isolation and characterisation of α2-chimaerin cDNA encoding a p21rac GTPase-activating protein with an SH2 domain

Sin, Wun Chey; (1994) Isolation and characterisation of α2-chimaerin cDNA encoding a p21rac GTPase-activating protein with an SH2 domain. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

n-Chimaerin is a neuronal GTPase-activating protein (GAP) for the small GTP-binding protein p21rac. In this study, a variant cDNA for α-chimaerin encoding a src homology 2 (SH2) domain at the N-terminal end of the sequence, in addition to the phorbol ester binding and GAP domains, was isolated from a human foetal retinal cDNA library. Except for the N-terminal, the α-chimaerin sequence is identical to that of n-chimaerin and is derived from an alternately spliced mRNA. The extreme 5' sequence was obtained by 5'RACE (rapid amplification of cDNA ends) of foetal and adult brain cDNA. The combined sequence was 2.1 kb with an open reading frame of 459 amino acids. Several 5'RACE products showed heterogeneity at the 5' end indicating possible alternate exon usage. A 4 kb cDNA, containing within it a sequence encoding the C-terminal half of the SH2 domain, was also isolated from the cDNA library. The rest of this cDNA has no counterpart in the database. SH2 domains bind selective phosphotyrosine-containing proteins. The full length α-chimaerin and its SH2 domain were expressed separately in E. coli as glutathione-S-transferase (GST) fusion proteins and the recombinant proteins were used in in vitro binding assays to identify potential α-SH2 interacting proteins. Rat pheochromocytoma (PC12) cells have been widely used as an in vitro model of neuronal differentiation. PC12 cells were stimulated with various growth factors including nerve growth factor (NGF) and fibroblast growth factor (FGF). GST/α-SH2 fusion protein specifically bound several 32P-labelled proteins (38 kDa, 45 kDa, 60 kDa, 84 kDa) whose phosphorylation was slightly increased in growth factor stimulated PC12 cells. GST/α-SH2 did not appear to bind EGF receptor overexpressed in A431 cells. Kinase activity was found to be associated with the α-SH2 binding proteins, which phosphorylated the SH2 domain in vitro at serine and threonine residues. The presence of an SH2 domain in α-chimaerin indicates that it is likely to interact with regulatory cellular components distinct from those of n-chimaerin in signalling pathways.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Isolation and characterisation of α2-chimaerin cDNA encoding a p21rac GTPase-activating protein with an SH2 domain
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10103084
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