Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin Dbinding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 Å resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to ''clamp'' onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-Å 2 intermolecular interface involve a mixture of hydrophobic, electrostatic, and solventmediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.
GABA B receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA B1 and GABA B2 subunits, neither of which is capable of producing functional GABA B receptors on homomeric expression. GABA B1, although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA B2 is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA B1 and GABA B2 subunits to explore further GABA B receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA B1 results in plasma membrane expression without the production of a functional GABA B receptor. However, coexpression of this truncated GABA B1 subunit with either GABA B2 or a truncated GABA B2 subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA B1 to GABA B2 leads to the ER retention of the GABA B2 subunit when expressed alone. These results indicate that the C terminal of GABA B1 mediates the ER retention of this protein and that neither of the C-terminal tails of GABA B1 or GABA B2 is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA B1 is capable of producing GABA-binding sites, GABA B2 is of central importance in the functional coupling of heteromeric GABA B receptors to G-proteins and the subsequent activation of effector systems.
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