Abstract
A novel synapse-associated protein, SAP90, accumulates around the axon hillock of Purkinje cells in rat cerebellum. By immuno-electron microscopy, SAP90 has been localized to the presynaptic termini of basket cells forming inhibitory, gamma-aminobutyric acid (GABA)ergic synapses onto Purkinje cell axon hillocks. The amino acid sequence for SAP90 has been deduced from the nucleotide sequence of a series of overlapping cDNA clones. SAP90 is related to the gene product encoded by the Drosophila tumor suppressor gene dlg-A. SAP90 and the dlg-A product share an overall sequence identity of 54%. Three distinct domains can be identified: (i) a potential cytoskeletal region consisting of three repeats of 90 amino acids in length, (ii) a domain with similarity to SH3, a putative regulatory motif found in the src family of non-receptor protein tyrosine kinases and several proteins associated with the cortical cytoskeleton, and (iii) a carboxyl-terminal domain homologous to yeast guanylate kinase. These features suggest a possible role for SAP90 in a guanine nucleotide-mediated signal transduction pathway at a subset of GABAergic synapses in the rat cerebellum.
Highlights
Utilizing a combined immunological and molecular approach, we have identified a number of novel and known components in synaptic junctional preparations
In the present study we describe the cloning ofcDNA encoding synapse-associated protein (SAP)’ 90, a protein found enriched in synaptic junctional preparations and located at the presynaptic termini of a subset of inhibitory synapses in the ratcerebellum
Clones encoding potential synapse-associated proteins (SAPS) phenylmethylsulfonyl fluoride; lane 2, 30,000 X g pellet of membrane were isolated with a rabbit fied synaptic bpjyuonslyccctrielooennnaailnl gapnrateripasaet rraubtmriaoingn.eXnAgertlalrtaeebdxbpairtgeasasinniotsinbt oalidbpyru,arriay-ffinetd1-rx.a0atcnrMtadecdTte,raiiinnmsd,lmaapnuneenldloe11bt.e%lTdoTthasritositf1om5Pn03e,Xm000-br01ar0atX0nbegr(alwpianeinltelhseu4t1b).wc,0e1alM%slutTlhaSrreDinsfrS,aps(celHtaqinou8ene.0ns5t)(i(.1ala6Pllnyapenge3ex)ols,f ity-purified using the fusion protein encoded by the X clone each) stained with the MA2d antibody ( d ) or with a synaptophysin
Summary
Antibody RA2d was affinity-purified from this antiserum using the Xgtll-expressed P-galactosidaseclone 2d fusion protein [13]. Antibody MA2dwas affinity-purified from a mouse polyclonal antiserum generated against glutathione S transferase-clone 2d fusion protein, constructed by cloning the 2d cDNA into the pGEX-2vTector (Pharmacia). Affinity-purified antibodies were isolated with fusion-proteins immobilized on nitrocellulose filters at 2 pg/cm2.
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