Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin.
The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein alpha-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of alpha-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.
Böckers
T M
TM
Arbeitsgruppe Molekulare Neurobiologie, Institut für Anatomie, Westfälische Wilhelms-Universität, 48149 Münster, Germany,.
Mameza
M G
MG
Kreutz
M R
MR
Bockmann
J
J
Weise
C
C
Buck
F
F
Richter
D
D
Gundelfinger
E D
ED
Kreienkamp
H J
HJ
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000648:ultrastructure*; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D006624:Hippocampus / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013569:Synapses / Q000737:chemistry / Q000648:ultrastructure; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains; D013049:Spectrin
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D013049:Spectrin; C120531:Shank1 protein, rat; C120532:Shank3 protein, rat