Signal Regulatory Proteins (SIRPS) Are Secreted Presynaptic Organizing Molecules

Hisashi Umemori,Joshua R Sanes

doi:10.1074/jbc.m805729200

Hisashi Umemori, Joshua R Sanes

Open Access

https://doi.org/10.1074/jbc.m805729200

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Abstract

Formation of chemical synapses requires exchange of organizing signals between the synaptic partners. Using synaptic vesicle aggregation in cultured neurons as a marker of presynaptic differentiation, we purified candidate presynaptic organizers from mouse brain. A major bioactive species was the extracellular domain of signal regulatory protein alpha (SIRP-alpha), a transmembrane immunoglobulin superfamily member concentrated at synapses. The extracellular domain of SIRP-alpha is cleaved and shed in a developmentally regulated manner. The presynaptic organizing activity of SIRP-alpha is mediated in part by CD47. SIRP-alpha homologues, SIRP-beta and -gamma also have synaptic vesicle clustering activity. The effects of SIRP-alpha are distinct from those of another presynaptic organizer, FGF22: the two proteins induced vesicle clusters of different sizes, differed in their ability to promote neurite branching, and acted through different receptors and signaling pathways. SIRP family proteins may act together with other organizing molecules to pattern synapses.

Highlights

Pre- and postsynaptic specializations form in precise apposition to each other at sites where axons contact specific target cells
We showed that fibroblast growth factor (FGF)2 22 and its close relatives, FGFs 7 and 10, promote differentiation of neuromuscular and cerebellar synapses [7, 10]
Most of these studies focused on intracellular signals transduced by SIRP-␣ when it is engaged by its ligand, CD47/integrin-associated protein [16]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Motoneurons were prepared and cultured as described [7, 10]. Briefly, cells were dissociated from E5 chick spinal cord with trypsin. Purification and Identification of Synaptic Organizing Molecules—Extracts were prepared from the forebrains of ϳ100 P7 mouse pups (C57/BL6 strain) as described [7] by homogenizing in a Tris buffer (pH 7.5) containing 400 mM NaCl. The extract was centrifuged at 20,000 ϫ g for 30 min and the supernatant was dialyzed against 10 mM Tris (pH 8.0), applied to a DEAE-Sepharose FF column, and eluted with a 0 –500 mM linear NaCl gradient. Soluble SIRP proteins were produced by transiently transfecting extracellular domain plasmids into COS cells, and purifying secreted SIRP proteins from culture media by affinity chromatography on anti-FLAG-agarose (Sigma). Immunoprecipitation—C2C12 culture media (from myoblasts and myotubes) were collected, precleared with 100 ␮l of Immobilized Protein-L (Pierce), and incubated with 2.5 ␮g of anti-SIRP-␣ extracellular domain antibody at 4 °C overnight. Primers used were: 5Ј-ATA CGC AGA CCT GAA TGT GCC CAA-3Ј and 5Ј-TGG CCA CTC CAT GTA GGA CAA GAA-3Ј for SIRP-␣, and 5Ј-AGA GCT GAA GGT GAT CCA ACC TGT-3Ј and 3Ј-ACC CGA ATC AGC AGG AGT GAC ATT-3Ј for SIRP-␤1

RESULTS

Purification of presynaptic organizing molecules from brain extracts

Total activity Purification