Our laboratory has discovered a structural and immunological analogue of rbc protein 4.1 in mammalian brain tissue. This brain protein 4.1 analogue (termed amelin) is distinct from synapsin I (another 4.1 analogue), and has the ability to bind brain spectrin in a solid phase assay. In the studies described in this proposal, the content of amelin in mouse brain will be quantitated with an immunodot assay, and compared to brain spectrin isoform content. The subcellular location of amelin in neural cells will be determined by immunoelectron microscopy utilizing the immunogold technology. Amelin's interaction with purified brain spectrin isoforms in solution will be tested by a bead filtration assay, and the effect of amelin phosphorylation upon the affinity and stoichiometry ascertained. In addition, we will localize the binding site for amelin along the brain spectrin molecule by rotary shadowing and electron microscopy of the complex. The ability of amelin to stimulate the brain spectrin isoform-F-actin interaction will be determined by sedimentation assays and low shear viscometry measurements. Again the effect of amelin phosphorylation upon this function will be ascertained. Finally the sequence of amelin will be determined, and compared to previously published sequences for rbc 4.1 and synapsin I. cDNA representing the coding region of the amelin gene will be isolated and sequenced by the dideoxy nucleotide chain termination method. These studies will give us a comprehensive knowledge of the structure, location, and function of this newly discovered neural membrane skeletal protein.
