Functional domains of neuromodulin, and, The interaction of calmodulin with target peptides

Calmodulin. To learn more about the molecular basis for calmodulin {dollar}cdot{dollar} target recognition, we have applied fluorescence spectroscopic methods and studied the interaction of calmodulin with the tryptophan residue in the calmodulin binding domains of skeletal muscle myosin light chain kinase, the plasma membrane calcium pump and an analog of the calmodulin binding domain of neuromodulin. Anisotropy and iodide quenching measurements indicated the tryptophan residue of each peptide was inserted into a hydrophobic cleft in calmodulin. Using Forster energy transfer measurements, the insertion site was localized to the C-terminal lobe of calmodulin. The orientation of the calcium pump peptide was determined by interchanging tryptophan-4 and phenylalanine-21. These data indicated that tryptophan-4 and phenylalanine-21 of the calcium pump peptide simultaneously interact with the hydrophobic clefts in the C- and N-terminal lobes of CaM respectively.; Neuromodulin. Neuromodulin (GAP-43) is a neural specific calmodulin binding protein which serves as a major presynaptic substrate for protein kinase C. To characterize the calmodulin binding domain of neuromodulin, wild type and mutant neuromodulins were expressed in bacteria, an in vitro transcription and translation system, or in cultured cells. These studies indicated that residues 39-56 are required for calmodulin binding. Substitution of serine-41 within this domain abolished protein kinase C phosphorylation of neuromodulin both in vitro and in vivo. Protein kinase C phosphorylation of neuromodulin reduces its affinity for calmodulin and was mimicked by the substitution of serine-41 with an acidic amino acid.; To study the association of neuromodulin with membranes and its targeting to growth cones, wild type, mutant and neuromodulin-reporter gene fusion constructs were expressed in cultured cells. These studies demonstrated that neuromodulin is anchored to membranes through palmitylation of its N-terminal cysteine residues. Palmitylation was not required for the rapid phosphorylation of neuromodulin by protein kinase C in vivo. However, these cysteine residues were required for the efficient accumulation of a neuromodulin-{dollar}beta{dollar}-galactosidase fusion protein in the growth cones of cultured neurons. Further analysis indicated that the first twenty amino acid residues of neuromodulin were sufficient for membrane targeting.