In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe 87 and Phe 88 have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca 2؉ /Zn 2؉ stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe 87 and Phe 88 residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val135 is reverted by ectopic expression of the calcium-and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca 2؉-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca 2؉ -dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca 2؉ -and Zn 2؉ -dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn 2؉ equivalents/mol S100B enhances Ca 2؉-dependent S100B-AHNAK interaction and that the effect of Zn 2؉ relies on Zn 2؉ -dependent regulation of S100B affinity for Ca 2؉. Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca 2؉ homeostasis.

The Zn 2؉ -and Ca 2؉ -binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn 2؉ and Ca 2؉ . Zn 2؉ binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca 2؉ -dependent manner within newly formed vesicle-like structures. Our data identify IQ-GAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn 2؉ /Ca 2؉ -dependent relocation of S100B. S100B is a member of the S100 family of proteins containing two EF-hand-type calcium-binding domains (1). This protein interacts not only with Ca 2ϩ but also with Zn 2ϩ ions, binding Zn 2ϩ ions with an affinity in the nanomolar range (2). The capacity of S100B to bind and release Zn 2ϩ suggests that Zn 2ϩ may not only play a structural role but might also be involved, together with Ca 2ϩ , in concerted regulation of S100B function. The S100B protein is naturally highly expressed in the vertebrate nervous system, where it is present in astrocytes and Schwann cells (3). In the adult central nervous system, the S100B protein is present in the nuclei and cytoplasm of astrocytes and accumulates in the astrocytic dendrites in the perivascular processes (4). Studies in different laboratories suggest a variety of intracellular regulations by S100B, including negative cell growth regulation (5), cell structure (6), and calcium homeostasis (7). The S100B protein is also secreted from astrocytes and has extracellular functions (8). Extracellular S100B acts as a modulator of neuronal synaptic plasticity (9). Although nanomolar quantities have beneficial neurotrophic effects on nerve cells, high levels of this protein have been implicated in glia activation and could contribute to the development of brain pathology as observed in Down's syndrome and Alzheimer's disease (10). The recent observation that S100B triggers activation of the pro-inflammatory cell surface receptor receptor for advanced glycation end products has shed more light on its extracellular function (11). In cultured human astrocytoma U87 cells, S100B secretion is dependent on relocation of S100B toward vesicle-like structures at the periphery of the cells and is regulated by Ca 2ϩ and Zn 2ϩ (12). S100B can also be secreted into the bloodstream and cerebrospinal fluid and is a biochemical marker of brain damage or dysfunction in acute and chronic diseases (13,14). A relationship between S100B accumulation in the astrocy...