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Shank1, Shank2, and Shank3 constitute a family of proteins that may function as molecular scaffolds in the postsynaptic density (PSD). Shank directly interacts with GKAP and Homer, thus potentially bridging the N-methyl-D-aspartate receptor-PSD-95-GKAP complex and the mGluR-Homer complex in synapses (Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, S., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569 -582; 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., and Worley, P. F. (1999) Neuron 23, 583-592). Shank contains multiple domains for protein-protein interaction including ankyrin repeats, an SH3 domain, a PSD-95/Dlg/ZO-1 domain, a sterile ␣ motif domain, and a proline-rich region. By characterizing Shank cDNA clones and RT-PCR products, we found that there are four sites for alternative splicing in Shank1 and another four sites in Shank2, some of which result in deletion of specific domains of the Shank protein. In addition, the expression of the splice variants is differentially regulated in different regions of rat brain during development. Immunoblot analysis of Shank proteins in rat brain using five different Shank antibodies reveals marked heterogeneity in size (120 -240 kDa) and differential spatiotemporal expression. Shank1 immunoreactivity is concentrated at excitatory synaptic sites in adult brain, and the punctate staining of Shank1 is seen in developing rat brains as early as postnatal day 7. These results suggest that alternative splicing in the Shank family may be a mechanism that regulates the molecular structure of Shank and the spectrum of Shank-interacting proteins in the PSDs of adult and developing brain.The mechanisms underlying the molecular assemblage of molecules at the synapse are not well understood. Recently, a number of novel anchoring/scaffold proteins that are associated with the PSD 1 have been isolated (1-5). In particular, PSD-95/ SAP90, GRIP/ABP, and Homer/Vesl have been reported to be putative anchoring proteins for NMDA, AMPA, and metabotropic glutamate receptors, respectively (6 -14). These anchoring proteins also interact with a variety of signaling and cytoskeletal proteins, thereby organizing a unique multiprotein complex for each glutamate receptor (15-21). One interesting question is whether there is any physical link between these specific glutamate receptor complexes and whether these links are regulated.Recently, a synaptic protein, termed Shank, that may bridge the NMDA receptor complex and the mGluR receptor complex has been isolated (22, 23). Shank is made of five domain/ regions that are likely involved in protein-protein interactions: ankyrin repeats, an SH3 domain, a PDZ domain, an SAM domain, and a proline-rich region. The PDZ domain of Shank directly interacts with the C-terminal QTRL motif of GKAP/ SAPAP/DAP-1 (24 -26), a protein that binds to the GK domain of the PSD-95 family of proteins 28), SAP97 (29),30), and SAP102 (12, 13)). The proli...

Stargazin is a transmembrane protein that interacts with AMPARs 1 and regulates their synaptic targeting (1, 2). The stargazer, a spontaneous mutant mouse (3) with defects in the stargazin gene (Cacng2) (4), displays an absence seizure (also known as petit-mal or spike-wave) and, as the name implies, a head-tossing movement, probably because of a defect in the vestibular system (3). In addition, stargazer mice develop an ataxic gait (3) and severe impairment in classical eye-blink conditioning (5), probably because of a cerebellar malfunction. Both mRNA and protein levels of brain-derived neurotrophic factor are selectively reduced in cerebellar granule cells of stargazer mice (5, 6). Stargazin, a protein with a calculated molecular mass of 36 kDa, contains four putative transmembrane domains and a cytosolic C terminus, and its primary structure is closely related to that of the ␥ subunits of voltagegated calcium channels (7-10). Stargazin (or ␥-2) associates with neuronal calcium channel subunits in vivo (11) and inhibits calcium channel activity by increasing steady-state inactivation (4,7,11,12).The functional association between stargazin and AMPAR was initially ascertained by the observation that postsynaptic AMPAR currents are selectively impaired in cerebellar granule cells of stargazer mice (13). A subsequent study revealed that stargazin mediates synaptic targeting of AMPARs by two distinct mechanisms (14). Stargazin initially interacts with AMPARs and assists their translocation to the extrasynaptic surface membrane. Next, the AMPAR-stargazin complex is targeted to synaptic sites by binding to PSD-95 and related PDZ proteins. In support of this hypothesis, a stargazin mutant lacking the last four residues (stargazin⌬C) rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14). However, little is known about whether the stargazin-mediated synaptic targeting of AMPARs is regulated and, if so, what these regulatory mechanisms involve.The C terminus of stargazin contains the end sequence RRT-TPV, which belongs to the class I PDZ-binding motif, (S/T)XV (S/T, Ser or Thr; X, any aa residue; V, hydrophobic residue) (15-17). Interestingly, the RRTT sequence of the C terminus additionally corresponds to the consensus sequence for phosphorylation by PKA, (R/K)(R/X)X(S/T), suggesting that Thr at the Ϫ2 position (RRTTPV, designated T321) is phosphorylated by PKA. The crystal structure of the PDZ3 domain of PSD-95 (class I) complexed with the C terminus of CRIPT, a PSD-95-binding protein that ends with the QTSV sequence (18), reveals that the Thr residue at the Ϫ2 position interacts with His-372 of PDZ3 (15). Specifically, the hydroxyl oxygen of the Thr forms a hydrogen bond with the N-3 nitrogen of His-372. Therefore, phosphorylation of T321 at the stargazin C terminus may weaken the interaction between stargazin and the PDZ domains of PSD-95. Consistently, earlier data demonstrate that phosphorylation of the Ser residue at the Ϫ2 position of Kir2.3 (an inward rectifie...