Regulation of AKAP-membrane interactions by calcium

The AKAP gravin is a scaffold for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of β₂-adrenergic receptors. Gravin binds to the receptor through well characterized protein-protein interactions. These interactions are facilitated ∼1000-fold when gravin is anchored to the cytoplasmic leaflet of the plasma membrane. Although the N-terminal region (∼550 residues) is highly negatively charged and probably natively unfolded, it could anchor gravin to the inner leaflet through hydrophobic insertion of its N-terminal myristate and electrostatic binding of three short positively charged domains (PCDs). Loss of the site of N-myristoylation was found to affect neither AKAP macroscopic localization nor AKAP function. Synthetic peptides corresponding to PCD1-3 bound in vitro to unilamellar phospholipid vesicles with high affinity, a binding reversed by calmodulin in the presence of Ca²⁺. In vivo gravin localization is regulated by intracellular Ca²⁺, a function mapping to the N terminus of the protein harboring PCD1, PCD2, and PCD3. Mutation of any two PCDs eliminates membrane association of the non-myristoylated gravin, the sensitivity to Ca²⁺/calmodulin, and the ability of this scaffold to catalyze receptor resensitization and recycling.