**5. Conclusion**

356 Biochemistry

Despite these advances, our understanding of the regulatory mechanisms of addicsin expression and the range of addicsin functions is far from complete. The elucidation of the regulatory mechanism of addicsin expression under basal and pathological conditions is essential for understanding the physiological and pathological roles of addicsin. For instance, while addicsin has consensus PKC phosphorylation sequences, it is unclear whether PKC actually phosphorylates addicsin and controls addicsin functions *in vivo*. It is also unknown whether or how PKC phosphorylation affects the interaction between addicsin and Arl6ip1. To overcome these challenges, it is crucial to clarify whether PKC phosphorylation sites of addicsin are physiologically controlled by PKC signaling and by which PKC isoforms. Furthermore, it remains controversial whether addicsin is an integral membrane protein. Our results strongly support the notion that addicsin is a membrane-associating protein with a soluble and membrane-localized form. Thus, it is important to clarify the different molecular features and functions of the soluble and

**4. Future research perspective** 

membrane-localized forms of addicsin.

Fig. 2. A scheme of the proposed physiological functions of addicsin

Second, *in vivo* functional studies are still needed to clarify the physiological and pathological functions of addicsin. Accumulating evidence suggests that addicsin participates in various physiological and pathological processes *in vivo*, but the molecular mechanisms controlling the selective interaction of addicsin with multiple targets, including receptors and transporters, are unknown. Furthermore, many reports demonstrate that the physiological and pathological roles of addicsin are observed when expression of addicsin is increased by various stresses, including oxidative and chemical stress. Thus, the production of animal models that overexpressed addicsin in a tissue- or region-specific manner may be useful to analyze addicsin functions in various tissues, including the brain. At present, no studies have been undertaken in tissues outside the brain, although addicsin is ubiquitously

expressed in kidney, heart, and liver (Butchbach et al., 2002; Ikemoto et al., 2002).

In this chapter, we argued that Arl6ip1 is a novel addicsin-interacting protein that indirectly promotes PKC-dependent, EAAC1-mediated glutamate uptake by inhibiting the interaction of addicsin with EAAC1 at the plasma membrane. Based on these findings, we proposed the regulatory model of EAAC1-mediated glutamate uptake illustrated in Fig. 3. In this model, EAAC1-mediated glutamate uptake activity can be negatively and positively regulated by PKC activity depending on dynamic modulation by addicsin complexes. Thus, the cellular dynamics of addicsin is a key element regulating EAAC1-mediated glutamate uptake. The study of addicsin is still in its infancy, but future findings on the physiological and pathophysiological functions of addicsin could greatly clarify the role of EAAC1 (and other proteins regulated by addicsin) in health and disease.

Fig. 3. A regulatory model of EAAC1-mediated glutamate uptake in C6BU-1 cells
