Screening an expression library with a ligand probe: isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein.
The use of cloning vectors that express inserted 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 ligand-binding sites might exist in fusion proteins. Specifically we used 125I-labeled calmodulin as a probe to screen a mouse brain lambda gt11 library. One clone, lambda ICM-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 experiments and deduction of the predicted higher-order structure from sequence data indicate the binding site is, or is within, a basic, amphiphilic alpha-helical domain composed of approximately 20 amino acids. lambda ICM-1 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 observations that suggest lambda ICM-1 corresponds to 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.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004251:DNA Transposable Elements; D005796:Genes; D005822:Genetic Vectors; D051379:Mice; D009928:Organ Specificity; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D002148:Calmodulin-Binding Proteins; D004251:DNA Transposable Elements; D012333:RNA, Messenger; D004247:DNA