Ca2+-Signaling Cycle of a Membrane-Docking C2 Domain

Abstract

The C2 domain is a Ca²⁺-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eukaryotic signal-transducing proteins, including cytosolic phospholipase A₂ (cPLA₂) of the vertebrate inflammation pathway. Intracellular Ca²⁺ signals recruit the C2 domain of cPLA₂ to cellular membranes where the enzymatic domain hydrolyzes specific lipids to release arachidonic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA₂ binds two Ca²⁺ ions with positive cooperativity, yielding a conformational change and membrane docking. When Ca²⁺ is removed, the two Ca²⁺ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca²⁺-binding sites become occluded in the membrane-bound complex such that Ca²⁺ binding and dissociation are slowed. Dissociation of the two Ca²⁺ ions from the membrane-bound domain is an ordered sequential process, and release of the domain from the membrane is simultaneous with dissociation of the second ion. Thus, the Ca²⁺-signaling cycle of the C2 domain passes through an active, membrane-bound state possessing two occluded Ca²⁺ ions, one of which is essential for maintenance of the protein−membrane complex.