Jin‐Hee Han

Competition between neurons is necessary for refining neural circuits during development and may be important for selecting the neurons that participate in encoding memories in the adult brain. To examine neuronal competition during memory formation, we conducted experiments with mice in which we manipulated the function of CREB (adenosine 3',5'-monophosphate response element-binding protein) in subsets of neurons. Changes in CREB function influenced the probability that individual lateral amygdala neurons were recruited into a fear memory trace. Our results suggest a competitive model underlying memory formation, in which eligible neurons are selected to participate in amemorytrace as a function of their relative CREB activity at the time of learning.

Memories are thought to be encoded by sparsely distributed groups of neurons. However, identifying the precise neurons supporting a given memory (the memory trace) has been a long-standing challenge. We have shown previously that lateral amygdala (LA) neurons with increased cyclic adenosine monophosphate response element-binding protein (CREB) are preferentially activated by fear memory expression, which suggests that they are selectively recruited into the memory trace. We used an inducible diphtheria-toxin strategy to specifically ablate these neurons. Selectively deleting neurons overexpressing CREB (but not a similar portion of random LA neurons) after learning blocked expression of that fear memory. The resulting memory loss was robust and persistent, which suggests that the memory was permanently erased. These results establish a causal link between a specific neuronal subpopulation and memory expression, thereby identifying critical neurons within the memory trace.

Prefrontal brain areas are implicated in the control of fear behavior. However, how prefrontal circuits control fear response to innate threat is poorly understood. Here, we show that the anterior cingulate cortex (ACC) and its input to the basolateral nucleus of amygdala (BLA) contribute to innate fear response to a predator odor in mice. Optogenetic inactivation of the ACC enhances freezing response to fox urine without affecting conditioned freezing. Conversely, ACC stimulation robustly inhibits both innate and conditioned freezing. Circuit tracing and slice patch recordings demonstrate a monosynaptic glutamatergic connectivity of ACC-BLA but no or very sparse ACC input to the central amygdala. Finally, our optogenetic manipulations of the ACC-BLA projection suggest its inhibitory control of innate freezing response to predator odors. Together, our results reveal the role of the ACC and its projection to BLA in innate fear response to olfactory threat stimulus.

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 (∼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 βPIX, focal adhesion kinase, liprin-α, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, βPIX, 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 βPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ. 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 (∼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 βPIX, focal adhesion kinase, liprin-α, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, βPIX, 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 βPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ. The active zone is a specialized presynaptic plasma membrane region where synaptic vesicles dock and fuse (1Landis D.M. J. Electron Microsc. Tech. 1988; 10: 129-151Google Scholar). The cytoskeletal matrix (cytomatrix) assembled at active zones (CAZ) 1The abbreviations used are: CAZ, cytoskeletal matrix assembled at active zones; GRKBD, G-protein-coupled receptor kinase-binding domain; GAP, GTPase-activating protein; ARF, ADP-ribosylation factor; SHD, Spa2 homology domain; GEF, guanine nucleotide exchange factor; FAK, focal adhesion kinase; EGFP, enhanced green fluorescent protein; aa, amino acid(s); CMV, cytomegalovirus; GST, glutathione S-transferase; PBS, paxillin-binding subdomain; GBD, GIT-binding domain; RU, resonance units; DIV, daysin vitro ; EM, electron microscopy; hemagglutinin is a complex proteinaceous structure implicated in organizing the site of neurotransmitter release (2Garner C.C. Kindler S. Gundelfinger E.D. Curr. Opin. Neurobiol. 2000; 10: 321-327Google Scholar, 3Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell. Mol. Life Sci. 2001; 58: 94-116Google Scholar). Recent studies have identified several core CAZ components involved in orchestrating the formation and functions of the CAZ: Piccolo/aczonin (4Cases-Langhoff C. Voss B. Garner A.M. Appeltauer U. Takei K. Kindler S. Veh R.W. De Camilli P. Gundelfinger E.D. Garner C.C. Eur. J. Cell Biol. 1996; 69: 214-223Google Scholar, 5Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Google Scholar, 6Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Google Scholar), Bassoon (7tom Dieck S. Sanmarti-Vila L. Langnaese K. Richter K. Kindler S. Soyke A. Wex H. Smalla K.H. Kampf U. Franzer J.T. Stumm M. Garner C.C. Gundelfinger E.D. J. Cell Biol. 1998; 142: 499-509Google Scholar), RIM (8Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Nature. 1997; 388: 593-598Google Scholar), Munc13 (9Brose N. Hofmann K. Hata Y. Sudhof T.C. J. Biol. Chem. 1995; 270: 25273-25280Google Scholar), and CAST/ERC (10Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Google Scholar, 11Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Google Scholar). Piccolo is a large (∼530 kDa) CAZ protein that is spatially restricted to active zones within the nerve terminal (4Cases-Langhoff C. Voss B. Garner A.M. Appeltauer U. Takei K. Kindler S. Veh R.W. De Camilli P. Gundelfinger E.D. Garner C.C. Eur. J. Cell Biol. 1996; 69: 214-223Google Scholar, 5Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Google Scholar, 6Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Google Scholar). Through its two zinc fingers, Piccolo interacts with the prenylated Rab acceptor 1 (5Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Google Scholar), a small (185 aa) soluble protein known to bind regulators of endo- and exocytosis, including Rab3, Rab5, and VAMP2/synaptobrevin II (12Martincic I. Peralta M.E. Ngsee J.K. J. Biol. Chem. 1997; 272: 26991-26998Google Scholar). A proline-rich sequence of Piccolo binds the actin cytoskeleton regulator profilin (6Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Google Scholar). In addition, the C2A domain of Piccolo mediates homodimerization and heterodimerization with RIM (13Fujimoto K. Shibasaki T. Yokoi N. Kashima Y. Matsumoto M. Sasaki T. Tajima N. Iwanaga T. Seino S. J. Biol. Chem. 2002; 277: 50497-50502Google Scholar). Intriguingly, Piccolo associates with an ∼80-nm dense core granulated vesicle (termed Piccolo transport vesicle) that contains other active zone components, suggesting that Piccolo and related active zone components are transported to nascent synapses as a preassembled package for the rapid and efficient formation of active zones (14Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Google Scholar). Although Piccolo's size, domain structure, and association with an active zone precursor vesicle suggest that it may be an important organizer of the CAZ, little is known about the mechanism by which this organization is carried out. GIT1 was originally isolated as a protein interacting with G-protein-coupled receptor kinases (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar). The GIT family of proteins contains two known members, GIT1/Cat-1/p95-APP1 and GIT2/Cat-2/PKL/p95-APP2/KIAA0148 (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar, 16Bagrodia S. Bailey D. Lenard Z. Hart M. Guan J.L. Premont R.T. Taylor S.J. Cerione R.A. J. Biol. Chem. 1999; 274: 22393-22400Google Scholar, 17Di Cesare A. Paris S. Albertinazzi C. Dariozzi S. Andersen J. Mann M. Longhi R. de Curtis I. Nat. Cell Biol. 2000; 2: 521-530Google Scholar, 18Turner C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar, R.T. Claing A. Vitale N. S.J. Lefkowitz R.J. J. Biol. Chem. 2000; Scholar, C.E. Brown M.C. Curr. Opin. Cell Biol. 2001; Scholar, Curr. Opin. Cell Biol. 2000; Scholar, Sci. 2000; 25: Scholar). GIT proteins a GTPase-activating protein domain for ADP-ribosylation small proteins implicated in the of membrane and the actin cytoskeleton P. B. Curr. Opin. Cell Biol. 1999; Scholar). In addition, GIT proteins various for protein including the Spa2 homology domain and the G-protein-coupled receptor kinase-binding domain GIT proteins of various membrane proteins (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar, A. S.J. M. J.K. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; and the of focal adhesion by interacting with the guanine nucleotide exchange factor βPIX, focal adhesion and the focal adhesion protein S. Bailey D. Lenard Z. Hart M. Guan J.L. Premont R.T. Taylor S.J. Cerione R.A. J. Biol. Chem. 1999; 274: 22393-22400Google Scholar, 18Turner C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar, E. L. Mol. Cell. Biol. 2000; Scholar). However, functions of GIT proteins, their including the (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar, R.T. Claing A. Vitale N. S.J. Lefkowitz R.J. J. Biol. Chem. 2000; Scholar), Here we report that Piccolo interacts with GIT and various GIT-associated GIT proteins form homo- and which GIT proteins to form a complex with Piccolo and βPIX. These results suggest that GIT proteins in the organization of the CAZ. A was as E. M. A. M. Nature. 1995; Scholar). and activity the GIT1 was by GIT1 (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google and with of Piccolo and the site of site of and in and was by amino and and the site of The of GIT1 and Piccolo site of and Piccolo, and GIT1 and GIT1 the site of was the site of Bassoon was by of and and GIT1 GIT1 and by with and site of GIT1 and with mutations in the SHD and by GIT1 and sites of protein the of Piccolo and GIT1 site of Piccolo and GIT1 was the site of protein GIT1 was A with and GIT1 and GIT1 the site of by the of GIT1 SHD and and and GIT1 and in (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar, R.T. Claing A. Vitale N. S.J. Lefkowitz R.J. J. Biol. Chem. 2000; Scholar). The GIT-binding domain of was site of with with at for the with proteins and for by with proteins by the The by with and and used as and Piccolo was and site of A resonance of was a by at different was at a of resonance was by a was used as The of the was by of at at was by of Piccolo and by of the with and and GIT1 was the site of GIT1 GIT1 and GIT1 by with and with and S. D. J. Biol. Chem. 2001; Scholar). of GIT1 with mutations in the SHD and a with and with at for Piccolo by protein In was as M. S. E. R. M. J. 1999; Scholar). the of was with and The was with Piccolo at for with protein for by of the with GIT1 Piccolo 1 and and proteins and of protein molecular of of in and 1 and at in an at for and the and by with by GIT1 as Piccolo in with The have GIT1 (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar), J. J. E. J. S. E. J. Biol. Chem. 2002; 277: Scholar), S. J. H. J. R. M. R. T. R. and E. In and S. D. J. Biol. Chem. 2001; Scholar). Piccolo as I. Garner C.C. Gundelfinger E.D. J. 2001; Scholar). was a of and The and as K. The and in the with and in vitro and with at for and with Piccolo and GIT1 by and by identified the interaction between Piccolo and GIT1 by of a the GIT1 as of the was a of Piccolo a GIT-binding domain in Piccolo was to the of the the C-terminal proline-rich region a region in GIT1 was as the SHD a domain known to the association of GIT1 with and E. L. Mol. Cell. Biol. 2000; Scholar). In to Piccolo with which the domain structure with GIT1 R.T. Claing A. Vitale N. S.J. Lefkowitz R.J. J. Biol. Chem. 2000; Scholar), as as a of that of the C-terminal contains the SHD R.T. Claing A. Vitale N. S.J. Lefkowitz R.J. J. Biol. Chem. 2000; 1 for the organization of In a region of Bassoon that to the Piccolo homology to the GIT-binding domain of Piccolo and not interact with not This that the GIT proteins interact with Piccolo and that Piccolo and Bassoon, two closely related CAZ proteins known to colocalize in synapses (5Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Google Scholar), may have of the interaction between Piccolo and GIT proteins by the and resonance A and with of GIT1 by of and by with proteins used in the by and proteins by and by with with and by and by with homology domain; homology the and of the interaction by resonance was the a at the the of and by an to the interaction of Piccolo with GIT proteins, we and with GIT1 A with the results not GIT1 the SHD that the SHD of GIT1 mediates its interaction with In addition, not proteins that Piccolo interacts with GIT1. with and and an protein with the which the SHD J. J. J. Mol. Cell. Biol. 1999; Scholar), as and not with the results 1 that Piccolo interacts with the GIT family The of by the results 1 This be to in the and that bind the and of the interaction between Piccolo and we resonance in to the the The at the the of a of and an of the of that the of is the of to is that the GIT1 SHD site for The of the interaction in this may in to the region of GIT1 a for Piccolo in the 1 and may be that of the by have in the GIT1 SHD that to be important for to Piccolo for Piccolo and GIT1 colocalize in of we cultured in vitro with the presynaptic of Piccolo (5Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Google Scholar), Piccolo was at GIT1 a Piccolo, and the was with a of small the several of the synaptic GIT1 proteins, with the Piccolo The GIT1 that not with Piccolo to be GIT1 proteins, as by and These results suggest that GIT1 proteins are to synaptic as as sites and that presynaptic GIT1 proteins colocalize with in a of interaction between Piccolo and GIT1 a A of with Piccolo and with and In not In Piccolo and Piccolo and GIT1 form a complex in brain, we of the of with Piccolo Piccolo and GIT1 and various proteins that are known to with including S. Bailey D. Lenard Z. Hart M. Guan J.L. Premont R.T. Taylor S.J. Cerione R.A. J. Biol. Chem. 1999; 274: 22393-22400Google Scholar, 18Turner C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar, E. L. Mol. Cell. Biol. 2000; Scholar), E. L. Mol. Cell. Biol. 2000; Scholar), C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar), and the M. E. M. C. M. R.J. M. Neuron. 2002; not and The multiple in the the of in the S. T. D. H. D. 2000; 272: E. L. J. Cell Sci. 2001; Scholar). with Piccolo not of the These results that Piccolo associates with GIT1 and proteins in The GIT1 SHD contains two conserved that are in in a we for to Piccolo βPIX. that of 1 the GIT1 its interaction with Piccolo and suggesting of the SHD are for to SHD This is with the of the interaction the resonance results sequence of the in the SHD of various and that are in and by of these amino important for Piccolo these in the of the GIT1 SHD and these mutations the GIT1 and in the we that the The other mutations in the of and for Intriguingly, of the mutations to the suggesting the of these mutations are to be by in the SHD structure, and that Piccolo and bind to the SHD of GIT1 by different mechanisms. of the mutations to GIT1 the the However, the of the other mutations the interaction This be to an enhanced between GIT1 and Piccolo in the of GIT1 1 to the SHD mutations of GIT1 with its SHD-binding βPIX, and in The in the GIT1 SHD the and the other mutations with in the of with the results However, these mutations not GIT1 with with the results Intriguingly, mutations of the GIT1 SHD association of GIT1 with FAK, protein that binds to the GIT1 SHD E. L. Mol. Cell. Biol. 2000; Scholar), GIT1 a complex with these results suggest that of GIT1 an important in to Piccolo, and that Piccolo, βPIX, and bind to the SHD of GIT1 by different mechanisms. multidomain proteins form E. A. M. Neuron. 1996; Scholar, S. E. B. C. J. R.J. M. Neuron. 1999; Scholar, H. P. I. D. J. 1999; Scholar, S. N. Y. Acad. Sci. 1999; Scholar). This organization is to be a mechanism for the of synaptic GIT1 is a multidomain protein at synaptic sites and it interacts with Piccolo, a active zone we GIT proteins form with and with with and that GIT proteins form homo- and not In the GIT1 and with GIT1 and the results and that GIT1 and form homo- and the region the we of GIT1 for their to form The C-terminal not the of GIT1 with GIT1 and in the that the mediates homo- and In the a protein the of GIT1 not GIT1 the and the GRKBD, not the the results The of GIT1 by as with that of the may that GIT1 proteins form that are for by the that the interaction between is these results that GIT proteins form homo- and heteromultimers through a direct interaction between in a tail-to-tail fashion. The of the is large and the contains several The region of the contains a domain with the of a at In addition, the C-terminal region of the contains paxillin-binding that is known to with C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar, E. L. Mol. Cell. Biol. 2000; Scholar). this we to the region the of the of the the and the region in between (termed and for to GIT1 and in the Intriguingly, the including the the the the with GIT1 and proteins the the the domain GIT1 These results that of the in the the of GIT1 we proteins of and the proteins a in their A and with the of molecular the of to a molecular of that of to to its the molecular of is the of to the of suggesting that GIT is to form The formation of a of a in between and is to be to and results and S. Bailey D. Lenard Z. Hart M. Guan J.L. Premont R.T. Taylor S.J. Cerione R.A. J. Biol. Chem. 1999; 274: 22393-22400Google Scholar, 18Turner C.E. Brown M.C. Perrotta J.A. Riedy M.C. Nikolopoulos S. S. J. Cell Biol. 1999; Scholar, E. L. Mol. Cell. Biol. 2000; that Piccolo, βPIX, and bind to the SHD of GIT1. of the functions of GIT multimerization is to GIT proteins to interact with multiple SHD-binding proteins as in A. this we to GIT1 that in multimerization of GIT1 that the domain of and the domain of and their multimerization by with and with of was in GIT1 not in GIT1 that the domain is for GIT The of domain GIT multimerization in the with that in be to the of the different the domain the different of the for the in and GIT1 used this to GIT1 multimerization is for the formation of a complex between Piccolo, and βPIX. with and with Piccolo was in the of not in the of GIT1 In GIT1 and GIT1 a with Piccolo that the of the complex in GIT1 not a of GIT1 to Piccolo βPIX. In to we that the of of the of by This that Piccolo and for to the GIT1 SHD and that the GIT1 SHD bind two different an of the of GIT we the in GRKBD, which by was to in the of proteins these results suggest that GIT multimerization is for the formation of the The molecular mechanisms the organization of the presynaptic CAZ are results that the CAZ protein Piccolo associates with GIT proteins as as various GIT-associated and signaling proteins, including liprin-α, βPIX, FAK, and paxillin. Intriguingly, Piccolo, βPIX, and bind to the SHD of GIT1 by different mechanisms. In addition, GIT proteins form homo- and heteromultimers through the GRKBD, and this multimerization enables GIT proteins to simultaneously interact with multiple SHD-binding proteins, including Piccolo and βPIX. These results suggest that GIT proteins, through multimerization and interaction with Piccolo, are involved in organizing the CAZ. that Piccolo associates with GIT proteins in vitro and in A of the interaction be their Piccolo is restricted to the active zone within the nerve terminal by (4Cases-Langhoff C. Voss B. Garner A.M. Appeltauer U. Takei K. Kindler S. Veh R.W. De Camilli P. Gundelfinger E.D. Garner C.C. Eur. J. Cell Biol. 1996; 69: 214-223Google Scholar, 6Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Google Scholar). that GIT1 are at the active zone in to their in the However, the GIT1 is not to synaptic sites. a to sites as by a of including in cultured and of GIT1 proteins to several the and in R. M. J. Cell Sci. 2002; Scholar). a for the of the interaction the in their be that Piccolo may be involved in the of GIT proteins to the CAZ. A this is that Piccolo and GIT1 may form a complex in the preassembled Piccolo transport vesicle and are the plasma membrane of nascent Zhai that Piccolo and other of the active zone as Bassoon and colocalize in in of cultured However, we that Piccolo and GIT1 a in of cultured we the between Piccolo and it that GIT proteins are with the preassembled Piccolo transport vesicle and with Piccolo the formation of the CAZ. GIT proteins various for protein as as an A of GIT proteins be to various GIT-associated proteins to the of the proteins that we have to form a complex with Piccolo a protein of is the multidomain protein C. L. A. M. J. 1995; Scholar, C. M. Hart A. M. J. Biol. Chem. 1998; Scholar). and of in presynaptic active zones M. Y. Nature. 1999; Scholar, N. J. R. H. D. Neuron. 2002; Scholar). In of the for its presynaptic associates with the receptor a regulator of and synaptic D. H. Cell. 1996; Scholar, T. S. Neuron. 2001; Scholar, C. T. Neuron. 2001; Scholar), and with the active zone protein which neurotransmitter release (8Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Nature. 1997; 388: 593-598Google Scholar, S. T. K. M. Y. Schmitz F. Sudhof T.C. Nature. 2002; Scholar). In addition, associates with a family of domain proteins that interacts with various membrane and signaling proteins, including acid the and and their M. S. E. R. M. J. 1999; Scholar, K. J. P. A. R. Neuron. 1999; Scholar, D. Z. T. J. Biol. Chem. 1999; 274: Scholar, R. H. J. Neuron. 1998; Scholar, B. D. X. P. H. Neuron. 2000; Scholar, H. R.J. Nature. 1997; Scholar, S. P. L. B. C. S. R. R.J. Neuron. 1998; Scholar). studies that liprin-α, and are at the presynaptic nerve terminal in to sites M. S. E. R. M. J. 1999; Scholar, M. E. M. C. M. R.J. M. Neuron. 2002; Scholar, H. P. I. D. J. 1999; S. N. Y. Acad. Sci. 1999; these results suggest that Piccolo and GIT proteins, in with and various proteins, including and in the organization of the CAZ. that the GIT family proteins form homo- and heteromultimers through the GIT multimerization is to the and of sites at the active Piccolo a complex with through GIT suggesting that GIT multimerization may the association of Piccolo with other SHD-binding proteins, including and In of the synaptic of βPIX, of of βPIX, in synaptic structure and protein D. Neuron. 2001; Scholar). is in and implicated in the of various signaling and proteins, including the family of kinases J.A. A. P. M. 1999; Scholar). and that are to the of Piccolo through GIT and may in the organization of presynaptic active Intriguingly, mutations in the GIT1 SHD different the interaction of GIT1 with Piccolo, βPIX, and suggesting that Piccolo, βPIX, and bind to the SHD of GIT1 by different mechanisms. Although of the of these these mutations may be for the in of these in GIT proteins may various presynaptic functions through the of small GTPases. GIT1 of various membrane proteins that are by the in a and (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar, A. S.J. M. J.K. Lefkowitz R.J. Premont R.T. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar). the of of by GIT1 an domain (15Premont R.T. Claing A. Vitale N. Freeman J.L. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Google Scholar). a of GIT1 N. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Premont R.T. J. Biol. Chem. 2000; Scholar), is various in that it actin cytoskeleton in various Curr. Opin. Cell Biol. 2000; Scholar, H. J. Cell Biol. 1996; Scholar, J. Z. H. J. Cell Sci. 1998; Scholar, H. J. Cell Biol. 1997; Scholar, D. C.C. S. J. Biol. Chem. 1998; Scholar, H. Mol. Cell. Biol. 2000; Scholar, Mol. Biol. Cell. 1998; Scholar). proteins are in and the site in cultured through and Nat. 2002; Scholar). and through and in Cesare A. Paris S. Albertinazzi C. Dariozzi S. Andersen J. Mann M. Longhi R. de Curtis I. Nat. Cell Biol. 2000; 2: 521-530Google Scholar). neurotransmitter release at U. H. N. J. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar). these results suggest that GIT proteins, through their may be involved in the of receptor actin cytoskeleton and neurotransmitter release at the active In results suggest that GIT proteins, through their multimerization and association with Piccolo, to the formation of a protein at the active The be to the GIT family proteins presynaptic organization and various GIT-associated signaling are to at for the of and at the of and for and at of and for the