PEPTIDES CONTAINING THE RERMS SEQUENCE OF AMYLOID BETA A4 PROTEIN-PRECURSOR BIND CELL-SURFACE AND PROMOTE NEURITE EXTENSION/

Amyloid beta/A4 protein precursor (APP) is secreted into medium by mos t cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we us ed a clonal CNS neuronal line, B103, which does not synthesize detecta ble levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-fre e defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-overproducing cells or by the bacteria-p roduced sAPP-695 (named KB75). A series of peptides having sequences b etween Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowt h of B103 eels. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, alt hough the concentration required for the neuritotropic activity was hi gher than that of KB75. Binding assay using I-125-labeled APP 17-mer p eptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demo nstrated specific and saturable cell-surface binding sites. The predic ted K-D value was 20 +/- 5 nM and the B-max value was 80 +/- 8 fmol/10 (6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed f or the binding. A bacteria-produced sAPP fragment lacking the active 1 7-mer sequence (named KB75 delta) did not compete with I-125-labeled 1 7-mer for binding or stimulate neurite extension. A peptide of sequenc e RMSQ (APP 330-333), which partially overlaps the active sequence RER MS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accu mulation of inositol polyphosphates, suggesting that the APP 17-mer ef fects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension throu gh cell-surface binding and that the domain containing the RERMS seque nce (APP 323-332) represents the active site responsible for this func tion.