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p45 chimaerin was purified from rat brain cytosol (excluding cerebellum) by a series of column chromatography and detected by the GAP overlay assay and by immunoreactivity to C-terminal-chimaerin antibodies (Fig. 7). The

primary S-sepharose fraction (eluted with 100-200 mM NaCI) was found to contain the bulk of the p45 chimaerin separated from the other GAPs. This S- sepharose fractionation resulted in 3-fold purification of p45 chimaerin (Table 1). This fraction containing p45 chimaerin and the cdc42Hs/rac1-binding proteins, was then subjected to Q-sepharose chromatography; two peak fractions (100-300 mM NaCI) were pooled and applied to a zinc chelating sepharose. The Q-sepharose column allowed another 7-fold purification of p45 chimaerin (Table 1). The zinc chelating column removed remaining GAPs and separated the cdc42Hs/rac1 binding proteins from p45 chimaerin, and resulted in a 2-fold purification. The p45 chimaerin-containing fraction (20-50 mM imidazole) was then loaded onto a phenyl sepharose column, resulting in another 2-fold purification. A 30 kDa immunoreactive band still copurified with p45 chimaerin on this column. However on the hydroxylapatite column, the p30 immunoreactive band was removed from p45 chimaerin. After the Zinc chelating FPLC column and another Q-sepharose column, p45 chimaerin was found to be purified approximately 400-fold (Table 1).

Peptide sequence analvsis of p45 chimaerin

Sequence analysis of p45 chimaerin in the final Q-sepharose fraction, removed after SDS-PAGE by transfer to a PVDF membrane, revealed a blocked N-terminus. This protein was thus not derived by proteolysis of a larger GAP. Two tryptic peptides each differed by one amino acid from sequences 18- 29 and 430-439 of human oc2-chimaerin predicted from the cDNA (Fig. 9 and 10A). The C-terminal peptide sequence was identical to the predicted rat sequence. Thus, p45 chimaerin purified from rat brain likely corresponds to o2- chimaerin.

p45 chimaerin as a candidate for a2-chimaerin

a2-chimaerin cDNA encodes a putative product of approximately 50 kDa (Hall et al, 1993). p45 chimaerin comigrated with recombinant a2-chimaerin on SDS-gel electrophoresis (Fig. 14B) and both were immunoreactive to 0 - terminal-chimaerin antibodies. The brain distribution of p45 chimaerin correlated

with mRNA expression of a2-chimaerin on in situ hybridization. a2-chimaerin mRNA is widely distributed with highest levels in cortex and hippocampus, and barely detected in adult cerebellar granule neurons (Hall et al, 1993). In the cerebellum, a2-chimaerin mRNA is detected in embryonic brain, but decreased after 15 days. o2-chimaerin mRNA expression in brain and testis (Hall et al, 1993) also correlates with the tissue distribution of p45 chimaerin (Manser et al, 1992).

q2-chimaerin mRNA encodes a splice variant of chimaerin with a N-terminal SH2 domain

Four a2-chimaerin cDNAs have been isolated from screening of the human retinal and hippocampal libraries (Hall et al, 1993). a l -chimaerin cDNA encodes a zinc/phorbol ester receptor and a p21 rac GAP (Hall et al, 1990; Lim et al, 1992). a2-chi maeri n is a splice variant of a1-chimaerin with an additional N-terminal SH2 domain and both mRNAs were 2.2 kb (Hall et al, 1993). The N-terminal 58 amino acid sequence in a1-chimaerin is absent in a2-chimaerin, instead a2-chimaerin has an additional 183 amino acids before the zinc/phorbol ester binding and GAP domains. a2-chimaerin share similar mRNA sequence to a1-chimaerin from nucleotide 663 of a1-chimaerin onwards to the 3 ’ end. The a2-chimaerin SH2 domain shares 40% amino acid sequence homology with the SH2 domains in rasGAP, Abl and p60c-src kinase (see Fig. 47). The SH2 domains in other proteins has a conserved tryptophan residue at the start which is absent in o2-chimaerin SH2 domain. The conserved arginine residues and the (F/Y)L(I/V)RES phosphotyrosine-binding sequence are however present in o2-chimaerin SH2 domain. Thus o2-chimaerin is a multi-functional protein with 3 different domains. Interestingly, a2-chimaerin shares two similar domains with two other proteins; vav (SH2 and phorbol ester binding domain) and PI3- kinase (SH2 and GAP domain). To date, o2-chimaehn is the only other reported SH2-contain|ng GAP for the p21s, beside pi 20 rasGAP (Vogel et al, 1988; Trahey et al, 1988). RasGAP associates with receptors and cytoplasmic proteins in signalling complexes via the SH2 domains (Ellis et al, 1990). The chimaerin SH2 domain binds specific phosphoproteins (of molecular mass 120

« 2 -chimaerin c-abl GAP-N GAP-C Src p85a -N p85a-C « 2 -chimaerin c-abl GAP-N GAP-C Src p85a-N p85a-C SGIN-- QAGKS-G.SYtIRE G # - PENPF ETTK- s # - LLRGK--RE LY-Y- RVYHY INTAS RFNT- IIAM- YYIGG -LSD GGRYY SO FVLSFLSQMN ^YFgTSEN SVSDFDNAKGL - -G A C S V W D - -GNN -E - IQRjBjKICPT- PNNÇgMR NVKH YKI RKLDS|^FYI TS RTGF - - k l i k i f h r-Id g^ g f s d p l i -VKHCVINKT-ATGYGFAEPYNL - W E HHST IGYYSHVSCLLKGE IDHYRKEQI-VEG VAYYSKHADGLCH NHYRNESLAQYNP v l h y o h t s l v q h; ÛGLIT 2DGLIT LYPVAP KEPVPMQ -TNVCPTS & # v g Y t^^pVYAO

Fig. 47-Sequence comparison of a2-chimaerin SH2 domain. The highlighted areas represent conserved residues between chimaerin and other protein sequences.

kDa, 80 kDa, 60 kDa and 38 kDa) from growth factor stimulated PCI 2 cells in vitro (Hall et al, 1993). Phosphorylation of the above proteins is increased in NGF, aPGF and EGF-treated cells. Abl SH2 binds similarly sized phosphoproteins as a2-chimaerin, except the 38 kDa protein which is specific to chimaerin SH2. PLCy SH2 binds a similarly sized 38 kDa protein which is phosphorylated on tyrosine in NGF-treated PCI 2 cells (Ohmichi et al, 1992). The chimaerin SH2 does not bind to activated EGF receptor overexpressed in A431 cells (Hall et al, 1993). a2-chimaerin represents the first protein in which an SH2 domain is derived from alternative splicing, suggesting a different control mechanism for chimaerin isoforms which share similar catalytic and regulatory domains with different cellular targets.