Identification and characterization of a calmodulin-binding domain in Ral-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 chromatography. Based on internal peptide sequencing and its protein amino acid composition, this protein has been shown to be highly related, if not identical to, Ral-A, a Ras-related GTP-binding protein. This protein assignment is consistent with the findings that p28 binds [32P]GTP specifically and has low GTPase activity. In this study we describe the identification and characterization of a calmodulin-binding domain in Ral-A. The Ca2+-dependent interaction of p28 with calmodulin was first detected by a calmodulin affinity column. Gel overlay experiments of both p28 and recombinant Ral-A with biotinylated calmodulin provided strong evidence that Ral-A is a calmodulin-binding protein. A peptide of 18 residues (P18) with the sequence SKEKNGKKKRKSLAKRIR has been identified as a putative calmodulin-binding domain in Ral-A, because it comprises a basic/hydrophobic composition with the propensity to form an amphiphilic helix. P18 was synthesized, and its interaction with calmodulin by gel overlay was shown to be Ca2+-dependent. Circular dichroism analysis demonstrated that this interaction results in less alpha-helical content upon calmodulin complex formation. These results indicate that Ral-A is a calmodulin-binding protein, raising the possibility that it may be associated with Ca2+-dependent intracellular signaling pathways.
Wang
K L
KL
Department of Pharmacy, University of Sydney, New South Wales 2006, Australia.
Khan
M T
MT
Roufogalis
B D
BD
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D004910:Erythrocyte Membrane / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D012995:Solubility; D020725:ral GTP-Binding Proteins
D002147:Calmodulin; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins