CA2-SIGNALING CYCLE OF A MEMBRANE-DOCKING C2 DOMAIN()

The C2 domain is a Ca2+-dependent, membrane-targeting motif originally discovered in protein kinase C and recently identified in numerous eu karyotic signal-transducing proteins, including cytosolic phospholipas e A(2) (cPLA(2)) of the vertebrate inflammation pathway. Intracellular Ca2+ signals recruit the C2 domain of cPLA(2) to cellular membranes w here the enzymatic domain hydrolyzes specific lipids to release arachi donic acid, thereby initiating the inflammatory response. Equilibrium binding and stopped-flow kinetic experiments reveal that the C2 domain of human cPLA(2) binds two Ca2+ ions with positive cooperativity, yie lding a conformational change and membrane docking. When Ca2+ is remov ed, the two Ca2+ ions dissociate rapidly and virtually simultaneously from the isolated domain in solution. In contrast, the Ca2+-binding si tes become occluded in the membrane-bound complex such that Ca2+ bindi ng and dissociation are slowed. Dissociation of the two Ca2+ ions from the membrane-bound domain is an ordered sequential process, and relea se of the domain from the membrane is simultaneous with dissociation o f the second ion. Thus, the Ca2+-signaling cycle of the C2 domain pass es through an active, membrane-bound state possessing two occluded Ca2 + ions, one of which is essential for maintenance of the protein-membr ane complex.