Expression, Characterization, and Ligand Studies Involving Domains of the Chick Cell Surface Protein Neogenin

Author: Miskevich, Frank

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Dreyer, William J.

Committee Members: Dreyer, William J.; Bjorkman, Pamela J.; Mayo, Stephen L.; Zinn, Kai George; Fraser, Scott E.

Option: Molecular Biology and Biochemistry

Abstract

The vertebrate retinotectal projection provides an excellent developmental system to study mechanisms and molecules involved in precisely patterning the nervous system. The retina sends out a single topographic projection which maps in a one to one correspondence in the tectum. This correspondence is brought about by the interplay of numerous factors, including electrical activity, extracellular signals, and the interaction of various signal cascades within the retinal ganglion cell.

Neogenin is an alternatively spliced transmembrane protein homologous to a number of genes involved in neurite outgrowth and pathfinding. Its developmental expression in the retina suggested that it could be involved in differentiation or signaling events during the period when optic fibers are making initial connections in the tectum. The aim of this study was to identify extracellular and cytoplasmic signals carried by neogenin.

The immunoglobulin domains of neogenin were heterologously expressed in the yeast Pichia pastoris and biochemically characterized. This protein was then used to generate monoclonal antibodies against various epitopes in these domains hopefully to identify function blocking or cross-species reactive antibodies.

The intracellular isoforms of neogenin were expressed and characterized in E. coli to identify proteins which interact with neogenin. Proteins of 200, 140, 110, and 55 kD were specifically labeled in brain lysates. Both neogenin isoforms react with the proteins in a calcium dependent fashion. Affinity chromatography, antibody co-precipitation, and expression library screening were then attempted to identify these labeled proteins.

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