IDENTIFICATION AND CHARACTERIZATION OF A CALMODULIN-BINDING DOMAIN INRAL-A, A RAS-RELATED GTP-BINDING PROTEIN PURIFIED FROM HUMAN ERYTHROCYTE-MEMBRANE

A 28-kDa protein (p28) has been purified from Triton X-100 extracts of human erythrocyte plasma membrane by calmodulin affinity chromatograp hy. Based on internal peptide sequencing and its protein amino acid co mposition, this protein has been shown to be highly related, if not id entical to, Ral-A, a Ras-related GTP-binding protein, This protein ass ignment is consistent with the findings that p28 binds [P-32]GTP speci fically and has low GTPase activity, In this study we describe the ide ntification and characterization of a calmodulin-binding domain in Ral -A. The Ca2+-dependent interaction of p28 with calmodulin was first de tected by a calmodulin affinity column, Gel overlay experiments of bot h p28 and recombinant Ral-A with biotinylated calmodulin provided stro ng evidence that Ral-A is a calmodulin-binding protein, A peptide of 1 8 residues (P18) with the sequence SKEKNGKKKRKSLAKRIR has been identif ied as a putative calmodulin-binding domain in Ral-A, because it compr ises a basic/hydrophobic composition with the propensity to form an am phiphilic helix, P18 was synthesized, and its interaction with calmodu lin by gel overlay was shown to be Ca2+-dependent, Circular dichroism analysis demonstrated that this interaction results in less alpha-heli cal content upon calmodulin complex formation, These results indicate that Ral-A is a calmodulin-binding protein, raising the possibility th at it may be associated with Ca2+-dependent intracellular signaling pa thways.