Screening an expression library wi and sequence of a cDNA correspon calmodulin-binding protein

The use of cloning vectors that express in- serted cDNA as fusion protein has led to the isolation of genes encoding a variety of eukaryotic proteins. In these instances antisera or monoclonal antibodies were used as probes to screen expression libraries. Since fusion proteins sometimes display biological activity reflective of the insert-specified portion, we tested the possibility that ligarid-binding sites might exist in fusion proteins. Specifically we used 125I-labeled calmodulin as a probe to screen a mouse brain kgtll library. One clone, XICM-1 isolated using this approach, produces fusion protein that binds calmodulin with high affinity (Kd, 3-10 nM) in a Ca2+-dependent manner. Molecular genetic mapping experi- ments and deduction of the predicted higher-order structure from sequence data indicate the binding site is, or is within, a basic, amphiphilic a-helical domain composed of =20 amino acids. XICM-I hybridizes with brain mRNA of 2.1 and 3.5 kb but not with mRNA from liver or kidney, suggesting possible restriction of the protein to brain. We discuss several obser- vations that suggest AICM-1 corresponds tb Ca2+/calmodulin- dependent protein kinase II, an enzyme that phosphorylates several neuronal proteins, some of which apparently play a role in synaptic function. Our results suggest certain types of ligands may be useful probes to isolate genes encoding various receptor proteins, particularly when the protein is very rare or when it is difficult to obtain antibodies suitable for screening libraries.