The Interaction of Phospholipase C-β3 with Shank2 Regulates mGluR-mediated Calcium Signal

Abstract

Phospholipase C-β isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-β isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-β3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-β3, but not other PLC-β isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-β3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-β3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-β3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses. Phospholipase C-β isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-β isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-β3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-β3, but not other PLC-β isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-β3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-β3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-β3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses. Phosphoinositide-specific phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; PSD, post-synaptic density; GST, glutathione S-transferase; TRITC, tetramethylrhodamine isothiocyanate; ACPD, N-1,3-cyclopentanedicarboxylic acid; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.1The abbreviations used are: PLC, phospholipase C; PSD, post-synaptic density; GST, glutathione S-transferase; TRITC, tetramethylrhodamine isothiocyanate; ACPD, N-1,3-cyclopentanedicarboxylic acid; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. plays a pivotal role in transmembrane signaling. In response to various extracellular stimuli, such as numerous hormones, neurotransmitters, metabolic products, and growth factors, PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), thereby generating messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (1.Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1197) Google Scholar, 2.Delmas P. Crest M. Brown D.A. Trends Neurosci. 2004; 27: 41-47Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Mammalian PLC isozymes have been classified into five types: PLC-β, PLC-γ, PLC-δ, PLC-ϵ, and PLC-ζ (3.Lopez I. Mak E.C. Ding J. Hamm H.E. Lomasney J.W. J. Biol. Chem. 2001; 276: 2758-2765Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 4.Song C. Hu C.D. Masago M. Kariyai K. Yamawaki-Kataoka Y. Shibatohge M. Wu D. Satoh T. Kataoka T. J. Biol. Chem. 2001; 276: 2752-2757Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 5.Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Royse B.L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar, 6.Coward K. Owen H. Poustka A.J. Hibbitt O. Tunwell R. Kubot Swann K. Parrington J. Biochem. Biophys. Res. Commun. 2004; 313: 894-901Crossref PubMed Scopus (14) Google Scholar). Immunohistological studies have shown that each isotype has a unique distribution pattern in the brain. PLC-β1 is highly expressed in the cerebral cortex and hippocampus, while PLC-β4 is enriched in the cerebellum. PLC-β3 is expressed throughout the brain (7.Ross C.A. MacCumber M.W. Glatt C.E. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2923-2927Crossref PubMed Scopus (115) Google Scholar, 8.Tanaka O Kondo H. Neurosci. Lett. 1994; 182: 17-20Crossref PubMed Scopus (69) Google Scholar). These findings suggest that each PLC-β isotype may have a distinct role. Amino acid sequence analysis has shown that PLC-β isotypes share a consensus sequence (X(S/T)X(V/L)-COOH) known as the postsynaptic density (PSD)-95/disc large/ZO-1 (PDZ) domain binding motif. PDZ domains exist in a large number of multifunctional proteins that mediate protein-protein interactions at highly specialized submembranous sites, such as the postsynaptic density and cellular junctions (9.Kornau H.C. Schenker L.T. Kenedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1611) Google Scholar, 10.Ponting C.P. Philips C. Davies K.E. Blake D.J. BioEssays. 1997; 19: 469-479Crossref PubMed Scopus (352) Google Scholar, 11.Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar). The postsynaptic density is a macroscopic submembranous multi-protein complex within the dendritic spines and contains a variety of scaffolding and signaling proteins. Shank/ProSAP, a core component of the PSD, is a multimodular protein (∼200 kDa) that contains various domains for protein interactions including ankyrin repeats, an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain (12.Boeckers T.M. Winter C. Smalla K.H. Kreutz M.R. Bockmann J. Seidenbecher C. Garner C.C. Gundelfinger E.D. Biochem. Biophys. Res. Commun. 1999; 264: 247-252Crossref PubMed Scopus (145) Google Scholar, 13.Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Crossref PubMed Google Scholar, 14.Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 15.Boeckers T.M. Bockmann J. Kreutz M.R. Gundelfinger E.D. J. Neurochem. 2002; 81: 903-910Crossref PubMed Scopus (267) Google Scholar). The PDZ domain of Shank interacts with the GKAP/SAPAP family of synaptic scaffold proteins. This demonstrates that the Shank family of multidomain proteins (Shank1/2/3) plays an important role in the organization and maintenance of the PSD (16.Kreienkamp H.J. Zitzer H. Gundelfinger E.D. Richter D. Bockers T.M. J. Biol. Chem. 2000; 275: 32387-32390Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The metabotropic glutamate receptor (mGluR) triggers intracellular signaling events by activating PLC-β through the Gαq family (17.Masu M. Tanabe Y. Tsuchida K. Shigemoto R. Nakanishi S. Nature. 1991; 349: 760-765Crossref PubMed Scopus (988) Google Scholar, 18.Abe T. Sugihara H. Nawa H. Shigemoto R. Mizuno N. Nakanishi S. J. Biol. Chem. 1992; 267: 13361-13368Abstract Full Text PDF PubMed Google Scholar). mGluR is known to be important in defining the efficacy of mGluR coupling to PLC-β (19.Pin J.P. Joly C. Heinemann S.F. Bockaert J. EMBO J. 1994; 13: 342-348Crossref PubMed Scopus (164) Google Scholar, 20.Flor P.J. Gomeza J. Tones M.A. Kuhn R. Pin J.P. Knopfel T. J. Neurochem. 1996; 15: 3970-3981Google Scholar). In addition, the Homer proteins, a family of adaptor proteins found at excitatory synapses, interact with mGluR and functionally link to the receptor for IP3 generated by PLC (21.Brakeman P.R. Lanahan A.A. O'Brien R. Roche K. Barnes C.A. Huganir R.L. Worley P.F. Nature. 1997; 386: 284-288Crossref PubMed Scopus (917) Google Scholar, 22.Xiao B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 23.Tu J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar). This finding led us to postulate that the Homer protein may take part in the signal pathway between membrane mGluR and PLC. In this study, we identified Shank2 as a PLC-β3-interacting protein. Shank2 specifically interacts with the C terminus of PLC-β3 but not with other isotypes. Furthermore, Homer 1b clusters the complexes of PLC-β3 and Shank2. Importantly, disrupting the PLC-β3-Shank2 interaction by injection with synthetic C-terminal heptapeptide of PLC-β3 reduced the mGluR-induced intracellular calcium signal. These results suggest that the complex of PLC-β3, Shank2 and Homer 1b may participate in organizing the molecules involved in PLC-β3-mediated signaling in the PSD. Yeast Two-hybrid System—Yeast two-hybrid screening was performed as described previously (24.Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Cellular Interactions in Development; A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). pBHA (LexA fusion vector) carrying DNA oligomers encoding C-terminal heptapeptides of PLC-β3 were used as the bait in the screening of a rat brain cDNA library in which the cDNAs were inserted into the activation domain of GAL4 in pGAD10 (Clontech, Palo Alto, CA). We screened ∼1.5 × 106 primary transformants for interacting proteins. Clones specifically interacting with the bait were identified by His growth and X-gal activity assays. The plasmids of positive clones were sequenced, and the sequences were compared with sequences in the data bank using the National Center for Biotechnology Information (NCBI) BLAST system. Plasmid Constructs—To express the glutathione S-transferase (GST) fusion form of the C-terminal region of PLC-β3 in Escherichia coli, the cDNA of C-terminal 337 amino acids was amplified by PCR with primers 5′-GCGAATTCCAGCAAGGCTCACAGCTC-3′ and 5′-GCCTCGAGTCAAAGCTGGGTGTTTTCC-3′. The resultant PCR product was inserted as an EcoRI/XhoI fragment into pGEX4T-1 (Amersham Biosciences). To express green fluorescent protein (GFP)-tagged PLC-β3 in mammalian cells, PCR products using appropriate primers were digested with EcoRI/SmaI and then ligated into pEGFP-C1. The cDNA of Shank2 was kindly provided by M. Sheng from the Massachusetts Institute of Technology (25.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google Scholar). To express Myc-tagged Shank2 in mammalian cells, the cDNAs of whole Shank2 and PDZ domain-deleted Shank2 amplified by PCR were digested with SmaI/SalI, and then inserted into EcoRV/XhoI-digested pCDNA3.1/Myc-HisA. The GST-fused PDZ domain of Shank2 was generated by subcloning the SmaI/XhoI insert of amino acids 37–140 into pGEX4T-3. The cDNA of Homer 1b was isolated from the rat brain cDNA library by PCR using the sense primers 5′-GCGAATTCATGGGGGAACAACCTATC-3′ and the anti-sense primers of 5′-ACGCGTCGACTCATTTAATCATGATTGCTG-3′. The GST-fused Homer 1b was generated by subcloning the EcoRI/SalI inserts into pGEX4T-1. We also constructed the HA-tagged Homer 1b gene by subcloning the inserts into the EcoRI/SalI sites of pCDNA3-HA, a HA epitope gene-containing plasmid. Expression in COS7 Cells and Coimmunoprecipitation—COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 50 international units/ml penicillin, 50 μg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2, 95% air incubator at 37 °C. The cDNAs for one of the PLC-β isotypes carrying an N-terminal FLAG-epitope tag, and either the wild-type or PDZ-deleted form of Shank2 with a C-terminal Myc epitope tag, were co-expressed in the cells by transient transfection using Lipofectamine (Invitrogen, Life Technologies, Inc.). After 36 h, the cells were in μg/ml and The were at × for at and of the were with to protein for at for The complexes were by at × with and by in were by The proteins were then to to binding was with 5% The were then with the appropriate for at which were and with for at in and using (Amersham Biosciences). the with the was in acid and into 50 of each was with of protein After at × for h, of was with A in of 50 for at °C. The were with of and proteins were by as including IP3 receptor S.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Expression and of proteins were expressed in the E. the appropriate cDNA Expression was by for at °C. the cells were and in The at × for at was with (Amersham for at and with cells the PLC-β isotypes or Shank2 were with and The were then with of the fusion proteins at a of for at °C. The protein complexes were by and with by in were by and and in COS7 cells were with a of appropriate using the Lipofectamine (Invitrogen, Life Technologies, the cells were in for at 37 °C. The cells were with and in serum in at for the cells were with or at °C. After with the cells were with either or After with the were and by cultured rat as described previously M. Y. K. A. M. Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In was isolated from rat and then cultured in with 10% After in the medium was with supplemented with of and cultured for the cultured cells were with with in for h, with A and and then with and for The were with (25.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (892) Google and rat 1b J.C. Xiao B. J.P. Lanahan A.A. K. M. D.J. Worley P.F. Neuron. 1998; 21: Full Text Full Text PDF PubMed Scopus Google for The were then with and with and for After the were with and then using a of C-terminal of PLC-β and to mGluR cells, were for A was into the cells a to a of The microinjection of with was with microinjection and used as a microinjection In for an a was to the the by using a were into the The of the intracellular calcium was mGluR a the intracellular calcium was in and at Cells were with for at After with the cells were for in the of to the To the of we with at the of the To we the at the of the and from the of To the of extracellular injection were in of was provided by the of an and the was The was to the with a The intracellular calcium was an with an The of the used in from to and were to and intracellular of injection to to of PLC-β3 with Shank by C-terminal of PLC-β3 in of in the rat brain in of were Shank was with In addition, various of the synthetic C-terminal heptapeptide of each PLC-β isozymes were to with PLC-β3 and Shank After for at were by with was performed with the PLC-β3 by and by calcium data are expressed as a analysis was using Institute A was as between cells were using of of the PLC-β3-interacting C-terminal heptapeptide of PLC-β3 was used as bait in the two-hybrid screening for proteins interacting with PLC-β3. positive from a rat brain cDNA library was and and found to the N-terminal amino acids of Shank2, which a PDZ The of the interaction between the C-terminal motif of PLC-β3 and the various PDZ domains, including Shank2 was using the two-hybrid His activity and the of the cells with the PLC-β3 motif and the PDZ of Shank2 were to of the PLC-β3 motif and the of a positive were by transformants of the PDZ domain of or These results demonstrate that the PLC-β3 specifically interacts with interacts specifically with the PDZ domain binding motif of of PLC-β3 in a between PLC-β3 and Shank2 in the interaction between PLC-β3 and Shank2, protein was used in a of COS7 with or C-terminal amino PDZ binding PLC-β3, but not the was by Shank2 expressed in COS7 cells was by the C-terminal 337 amino but not of PLC-β and Shank2 in PLC-β isotypes the PDZ binding motif at their C that may the to with the PDZ domain-containing proteins. To the interaction between Shank2 and each PLC-β using an was performed with of COS7 cells Shank2 and each of the PLC-β isotypes. Shank2 was in the with but not with of the other isotypes that Shank2 specifically interacts with PLC-β3. To this the of COS7 cells and were with shown in and PLC-β3 was with Shank2, the PLC-β3 and the PDZ domain-deleted form of Shank2 were not with Shank2 and PLC-β3, These results suggest that PLC-β3 interacts with the PDZ domain of Shank2 the C-terminal PDZ binding motif. demonstrate that PDZ domains to the C-terminal amino acids of the proteins M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar). To of the PLC-β3 C terminus that are for the Shank2 each of the amino acid of PLC-β3 were to The of COS7 cells each PLC-β3 and Shank2 were with an and the were then with an of to at the amino acid or to at the in a of Shank2 binding to PLC-β3. In the of the other to These results that the and of PLC-β3 are for the interaction with Shank2. PLC-β3 with Homer 1b Through was that and with the domain of the Homer family J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (876) Google and sequence analysis that Shank2 Homer binding To that Shank2 also with Homer and that Shank2 link PLC-β3 to Homer 1b, COS7 cells Shank2 and PLC-β3 were used in with of COS7 cells Shank2 and Homer 1b were used for with the C-terminal motif. shown in A and 1b PLC-β3 in the of Shank2. the C terminus Homer 1b in the of Shank2, that proteins form a To the complex of PLC-β3, Shank2, and Homer 1b, was performed COS7 cells proteins. In expressed cells, PLC-β3, Shank2, and Homer 1b were in clusters and In a PLC-β1 not with Shank2 and Homer 1b These results are with the of interaction between PLC-β1 and Shank2 in PLC-β3 or Shank2 were co-expressed with the other proteins, PLC-β3 not with Shank2. the other the wild-type and the PDZ-deleted form of Shank2 were with Homer 1b, with the finding that the Homer binding region in the proline-rich region of Shank2 J.C. Xiao B. J.P. Lanahan A.A. K. M. D.J. Worley P.F. Neuron. 1998; 21: Full Text Full Text PDF PubMed Scopus Google Scholar). We the complex of PLC-β3, Shank2, and using isolated with the of PLC-β3, Shank2, and Homer 1b were as a form in cells also results suggest that Shank2 the of a complex Homer 1b and PLC-β3. of in we the in of Shank2, PLC-β3, Homer 1b, and IP3 the of rat brain was with Shank2 PLC-β3, Homer 1b, and IP3 receptor were but not with In addition, to C-terminal heptapeptide of PLC-β3 with the PLC-β3-Shank2 we the synthetic C-terminal peptide of PLC-β3 the we Shank2 with of rat brain. The of PLC-β3 with Shank2 by synthetic PLC-β3 peptide in a the was by of C-terminal of other isozymes results demonstrate that PLC-β3 and Shank2 may be to synaptic sites and form a complex in brain. of of PLC-β3 C-terminal mGluR into PLC-β3 binding with Shank2, we the of this binding mGluR intracellular calcium signal. To we used cells, cells an intracellular calcium in response to a mGluR N-1,3-cyclopentanedicarboxylic acid has been that Shank may Homer with the complex in the postsynaptic density and a role in the mGluR signaling. a has important in the coupling of mGluR receptors to intracellular calcium we that the calcium to the mGluR be by disrupting To this we a synthetic C-terminal heptapeptide of PLC-β3 into the of the cells and the calcium signal. a we C-terminal heptapeptides of other PLC-β Cells with PLC-β3 peptide a markedly calcium signal with of other PLC-β isozymes and not in the calcium signaling. we cells at a and performed These results were in To the of an extracellular calcium we performed the microinjection These results demonstrate that microinjection of the C-terminal peptide of PLC-β3 the mGluR calcium in a but C-terminal of other isozymes have results demonstrate that the PLC-β3-Shank2 interaction may an important role in mGluR-mediated intracellular calcium In this we identified a interaction between the C terminus of PLC-β3 and the PDZ domain of Shank2. In of this the C-terminal amino acids of PLC-β3 are for the interaction with Shank2 and at the PLC-β3 C terminus the Shank2 interaction In addition, data that Shank2 forms a specific complex with PLC-β3, but not with other PLC-β the that other PLC-β also the PDZ domain binding motif at their C These results suggest that PLC-β may in signaling by interacting with distinct proteins. we found that specifically interacts with and the activation of PLC-β3 by the receptor K. Kim E. C. S.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). other isotypes of PLC-β interact with to Shank2, the performed in the that Shank2 specifically with PLC-β3, but not with the other isotypes. In Shank2 was in brain but was not that each of PDZ proteins may a role in PLC-β3-mediated signaling in has been known that proteins mediate the of receptors and signaling molecules and thereby signal S.H. M. Kim Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). the signaling through PLC-β3 may be in cells the interaction with proteins. of Shank2 have been shown to interact with the Homer domain-containing proteins J.C. Xiao B. Naisbitt S. Yuan J.P. Petralia R.S. Brakeman P. Doan A. Aakalu V.K. Lanahan A.A. Sheng M. Worley P.F. Neuron. 1999; 23: 583-592Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar). has been that Homer in a that the and of the C. K. K. Worley P.F. Y. Sheng M. J. Neurosci. 23: PubMed Google Scholar). Homer proteins exist in forms and are known to interact with group metabotropic glutamate receptors and the inositol 1,4,5-trisphosphate and may be involved in the calcium by a product of PLC activation B. Tu J.C. Petralia R.S. Yuan J.P. Doan A. Breder C.D. Ruggiero A. Lanahan A.A. Wenthold R.J. Worley P.F. Neuron. 1998; 21: 707-716Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, D.J. R.S. P. J. Biol. 86: PubMed Scopus Google Scholar). we that Shank2 may as a scaffold for G protein-coupled receptor signaling in the PSD by between the Homer family and PLC-β3. Shank2, and PLC-β3 a to form clusters are with Homer The using of Shank2 or PLC-β3 that PLC-β3 is into 1b complexes through PDZ previously Shank2 is enriched in the PSD S. Naisbitt S. J. Sheng M. Kim E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). PSD signaling events in neuronal cells mGluR signaling S. T. T. K. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google is that PLC-β3 may be involved in mGluR-mediated and Homer 1b calcium J.C. Xiao B. J.P. Lanahan A.A. K. M. D.J. Worley P.F. Neuron. 1998; 21: Full Text Full Text PDF PubMed Scopus Google Scholar). to the role of scaffolding proteins in receptors is H. Sheng M. Rev. Neurosci. 2001; PubMed Scopus Google Scholar). is known the scaffolding role in calcium signaling. In synapses, and other participate in the of signaling including the role for calcium signaling. In the study, we provided the for the role of the binding between Shank2 and PLC-β3 in mGluR-mediated calcium We not the of Shank2 PLC-β calcium response through signal molecules to the mGluR receptor or PLC-β activity is to be In PLC-β3 interacts with Shank2 the PDZ binding motif. The interaction of PLC-β3 with Shank2 that proteins signaling molecules to be involved in a specific signaling by to specific Homer 1b, a I forms clusters with PLC-β3, which is by Shank2. of the complex of Homer the signaling complexes in the PSD are This finding may us to a of the activation of PLC-β3 by in the PSD of neuronal