Domain Structure of the Shank Proteins

Three genes coding for Shank isoforms have been discovered in various mammalian species; all three forms, labeled Shank1-3, are expressed strongly in the central nervous system. In addition, individual isoforms are expressed at lower levels in peripheral tissues (Lim et al. 1999). All three Shank forms are characterized by a common series of protein interaction motifs or domains, i.e., a set of ankyrin repeats, followed by SH3- and PDZ-domains, a rather long proline-rich region and a C-terminal SAM domain (Fig. 1). The calculated molecular weights range from 130 to 230 kDa; in Western blot experiments, these forms migrate at apparent molecular weights ranging from 180 kDa to more than 250 kDa. This difference is probably due to a large proportion of unfolded protein regions within the 1,000-aa-long proline-rich region.

Western blot analysis also indicates the presence of multiple molecular forms of Shank proteins in which individual parts of the domain structure may be missing due to alternative splicing and alternative promoter usage (Lim et al. 1999; Boeckers et al. 1999a; see Fig. 1). Thus, the major form of Shank2 expressed in the brain does not contain the ankyrin and SH3 domains; the term "Shank" therefore does not really apply to this protein, and the alternative ProSAP makes indeed more sense. This form occurs due to a transcriptional start site within the long intron separating the genomic segments coding for SH3 and PDZ domains. A "full-length" form of Shank2 containing SH3 and ankyrin regions is expressed mostly in epithelial cells and has been labeled Shank2E (McWilliams et al. 2004).

In neurons, Shank proteins have been localized almost exclusively to the postsy-naptic specialization of excitatory synapses. Biochemical preparations indicate a very strong enrichment in the PSD, and Shank immunoreactivity is in fact one of the most reliable markers for the PSD (Boeckers 1999a; Naisbitt et al. 1999; Zitzer et al. 1999a). Quantitative analyses have led to an estimate of about 100 Shank molecules per mature PSD complex (Chen et al. 2005; Sugiyama et al. 2005). A detailed analysis by immunogold electron microscopy indicated that the majority of Shank is located deep within the PSD, at a position remote from the synaptic plasma membrane. In contrast, glutamate receptors and their directly associated scaffold proteins from the SAP/PSD-95 family are located at the surface of the PSD, in direct contact with the plasma membrane (Valtschanoff and Weinberg 2001). Shank proteins are therefore not considered to provide a direct scaffolding function for transmitter receptors, but rather work indirectly by connecting different types of scaffold/receptor complexes, leading to the concept of a "master" or "higher order" scaffold. This view is strongly supported by the different protein interactions in which Shank proteins are involved, as detailed below.

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