The GIT family of proteins forms multimers and associates with the presynaptic cytomatrix protein Piccolo.
The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (approximately 150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including betaPIX, focal adhesion kinase, liprin-alpha, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, betaPIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and betaPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.
Kim
Seho
S
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Ko
Jaewon
J
Shin
Hyewon
H
Lee
Jae-Ran
JR
Lim
Chunghun
C
Han
Jin-Hee
JH
Altrock
Wilko D
WD
Garner
Craig C
CC
Gundelfinger
Eckart D
ED
Premont
Richard T
RT
Kaang
Bong-Kiun
BK
Kim
Eunjoon
E
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000502:physiology; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013569:Synapses / Q000502:physiology / Q000648:ultrastructure; D014162:Transfection; D016335:Zinc Fingers
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C485647:GIT1 protein, human; C430213:GIT2 protein, human; D020690:GTPase-Activating Proteins; C485648:Git1 protein, rat; D046911:Macromolecular Substances; D009479:Neuropeptides; C498630:PCLO protein, human; C405514:Pclo protein, rat; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase